Enzyme Treatment of Foodstuffs for Celiac Sprue

ABSTRACT

Administering an effective dose of glutenase to a Celiac or dermatitis herpetiformis patient reduces levels of toxic gluten oligopeptides, thereby attenuating or eliminating the damaging effects of gluten.

This invention was made with Government support under contract DK063158awarded by the National Institutes of Health. The Government has certainrights in this invention.

BACKGROUND OF THE INVENTION

In 1953, it was first recognized that ingestion of gluten, a commondietary protein present in wheat, barley and rye causes disease insensitive individuals. Gluten is a complex mixture of glutamine- andproline-rich glutenin and prolamine molecules, which is thought to beresponsible for disease induction. Ingestion of such proteins bysensitive individuals produces flattening of the normally luxurious,rug-like, epithelial lining of the small intestine known to beresponsible for efficient and extensive terminal digestion of peptidesand other nutrients. Clinical symptoms of Celiac Sprue include fatigue,chronic diarrhea, malabsorption of nutrients, weight loss, abdominaldistension, anemia, as well as a substantially enhanced risk for thedevelopment of osteoporosis and intestinal malignancies (lymphoma andcarcinoma). The disease has an incidence of approximately 1 in 200 inEuropean populations.

A related disease is dermatitis herpetiformis, which is a chroniceruption characterized by clusters of intensely pruritic vesicles,papules, and urticaria-like lesions. IgA deposits occur in almost allnormal-appearing and perilesional skin. Asymptomatic gluten-sensitiveenteropathy is found in 75 to 90% of patients and in some of theirrelatives. Onset is usually gradual. Itching and burning are severe, andscratching often obscures the primary lesions with eczematization ofnearby skin, leading to an erroneous diagnosis of eczema. Strictadherence to a gluten-free diet for prolonged periods may control thedisease in some patients, obviating or reducing the requirement for drugtherapy. Dapsone, sulfapyridine and colchicines are sometimes prescribedfor relief of itching.

Celiac Sprue is generally considered to be an autoimmune disease and theantibodies found in the serum of the patients supports a theory of animmunological nature of the disease. Antibodies to tissuetransglutaminase (tTG) and gliadin appear in almost 100% of the patientswith active Celiac Sprue, and the presence of such antibodies,particularly of the IgA class, has been used in diagnosis of thedisease.

The large majority of patients express the HLA-DQ2 [DQ(a1*0501, b1*02)]and/or DQ8 [DQ(a1*0301, b1*0302)] molecules. It is believed thatintestinal damage is caused by interactions between specific gliadinoligopeptides and the HLA-DQ2 or DQ8 antigen, which in turn induceproliferation of T lymphocytes in the sub-epithelial layers. T helper 1cells and cytokines apparently play a major role in a local inflammatoryprocess leading to villus atrophy of the small intestine.

At the present time there is no good therapy for the disease, except tocompletely avoid all foods containing gluten. Although gluten withdrawalhas transformed the prognosis for children and substantially improved itfor adults, some people still die of the disease, mainly adults who hadsevere disease at the outset. An important cause of death islymphoreticular disease (especially intestinal lymphoma). It is notknown whether a gluten-free diet diminishes this risk. Apparent clinicalremission is often associated with histologic relapse that is detectedonly by review biopsies or by increased EMA titers.

Gluten is so widely used, for example in commercial soups, sauces, icecreams, hot dogs, and other foods, that patients need detailed lists offoodstuffs to avoid and expert advice from a dietitian familiar withceliac disease. Ingesting even small amounts of gluten may preventremission or induce relapse. Supplementary vitamins, minerals, andhematinics may also be required, depending on deficiency. A few patientsrespond poorly or not at all to gluten withdrawal, either because thediagnosis is incorrect or because the disease is refractory. In thelatter case, oral corticosteroids (e.g., prednisone 10 to 20 mg bid) mayinduce response.

In view of the serious and widespread nature of Celiac Sprue, improvedmethods of treating or ameliorating the effects of the disease areneeded. The present invention addresses such needs.

SUMMARY OF THE INVENTION

The present invention provides methods for treating the symptoms ofCeliac Sprue and/or dermatitis herpetiformis by decreasing the levels oftoxic gluten oligopeptides in foodstuffs, either prior to or afteringestion by a patient. The present invention relates to the discoverythat certain gluten oligopeptides resistant to cleavage by gastric andpancreatic enzymes, that the presence of such peptides results in toxiceffects, and that enzymatic treatment can remove such peptides and theirtoxic effects. By digestion with glutenases, these toxic oligopeptidesare cleaved into fragments, thereby preventing or relieving their toxiceffects in Celiac Sprue or dermatitis herpetiformis patients.

In one aspect of the invention, a foodstuff is treated with a glutenaseprior to consumption by the patient. In another aspect of the invention,a glutenase is administered to a patient and acts internally to destroythe toxic oligopeptides. In another aspect of the invention, arecombinant organism that produces a glutenase is administered to apatient. In another aspect of the invention, gene therapy is used toprovide the patient with a gene that expresses a glutenase that destroysthe toxic oligopeptides.

In one aspect of the invention, methods are provided for initialassessment of patients, and for monitoring patients during treatment. Ithas surprisingly been found that a high percentage of patients believedto be in remission were suffering from intestinal malabsorption andmalfunction. In some embodiments of the invention, the subject therapycomprises the steps of monitoring and/or diagnosis with assays forintestinal malabsorption and malfunction. Such monitoring also finds usein the evaluation of the therapeutic efficacy of clinical protocolsand/or formulations.

In one aspect, the invention provides methods for the administration ofenteric formulations of one or more glutenases, each of which may bepresent as a single agent or a combination of active agents. In anotheraspect of the invention, stabilized forms of glutenases are administeredto the patient, which stabilized forms are resistant to digestion in thestomach, e.g. to acidic conditions. Alternative methods ofadministration include genetic modification of patient cells, e.g.enterocytes, to express increased levels of peptidases capable ofcleaving immunogenic oligopeptides of gliadin; pretreatment of foodswith glutenases; the introduction of micro-organisms expressing suchpeptidases so as to transiently or permanently colonize the patientintestinal tract; and the like.

In another aspect, the invention provides pharmaceutical formulationscontaining one or more glutenases and a pharmaceutically acceptablecarrier. Such formulations include formulations in which the glutenaseis contained within an enteric coating that allows delivery of theactive agent to the intestine and formulations in which the activeagents are stabilized to resist digestion in acidic stomach conditions.The formulation may comprise one or more glutenases or a mixture or“cocktail” of agents having different activities.

In another aspect, the invention provides foodstuffs derived fromgluten-containing foods that have been treated to remove or to reduce tonon-toxic levels the gluten-derived oligopeptides that are toxic toCeliac Sprue patients, and methods for treating foods to hydrolyze toxicgluten oligopeptides. In other aspects, the invention providesrecombinant microorganisms useful in hydrolyzing the gluten-derivedoligopeptides that are toxic to Celiac Sprue patients from foodstuffs;methods for producing glutenases that digest the gluten-derivedoligopeptides that are toxic to Celiac Sprue patents; purifiedpreparations of the glutenases that digest the gluten-derivedoligopeptides that are toxic to Celiac Sprue patents; and recombinantvectors that code for the expression of glutenases that digest thegluten-derived oligopeptides that are toxic to Celiac Sprue patents.

These and other aspects and embodiments of the invention are describedin more detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B. Brush border membrane catalyzed digestion of theimmunodominant gliadin peptide. FIG. 1A: LC-MS traces of peptides asshown, after digestion with 27 ng/μl rat brush border membrane (BBM)protein for the indicated time. Reaction products were separated byreversed phase HPLC and detected by mass spectroscopy (ion countsm/z=300-2000 g/mol). The indicated peptide fragments were confirmed bycharacteristic tandem MS fragmentation patterns. The SEQ ID NO:2pyroQLQPFPQPQLPY peak corresponds to an N-terminally pyroglutaminatedspecies, which is generated during HPLC purification of the syntheticstarting material. FIG. 1B: Abundance of individual digestion productsas a function of time. The peptide fragments in FIG. 1A were quantifiedby integrating the corresponding MS peak area (m/z=300-2000 g/mol). Theresulting MS intensities are plotted as a function of digestion time(with BBM only). The digestion experiment was repeated in the presenceof exogenous DPP IV from Aspergillus fumigatus (Chemicon International,CA, 0.28 μU DPP IV/ng BBM protein) and analyzed as above (open bars).The relative abundance of different intermediates could be estimatedfrom the UV₂₈₀ traces and control experiments using authentic standards.The inserted scheme shows an interpretative diagram of the digestionpathways of SEQ ID NO:1) QLQPFPQPQLPY and its intermediates, the BBMpeptidases involved in each step, and the amino acid residues that arereleased. The preferred breakdown pathway is indicated in bold.APN=aminopeptidase N, CPP=carboxypeptidase P, DPP IV=dipeptidyldipeptidase IV.

FIG. 2A-2B. C-terminal digestion of the immunodominant gliadin peptideby brush border membrane. FIG. 2A: (SEQ ID NO:3) PQPQLPYPQPQLPY wasdigested by 27 ng/μl brush border membrane (BBM) protein preparationsfor the indicated time and analyzed as in FIG. 1A. The identity of thestarting material and the product (SEQ ID NO:4) PQPQLPYPQPQLP wascorroborated by MSMS fragmentation. The intrinsic mass intensities ofthe two peptides were identical, and the UV₂₈₀ extinction coefficient of(SEQ ID NO:4) PQPQLPYPQPQLP was half of the starting material inaccordance with the loss of one tyrosine. All other intermediates were1%. The scheme below shows the proposed BBM digestion pathway of (SEQ IDNO:3) PQPQLPYPQPQLPY with no observed N-terminal processing (crossedarrow) and the removal of the C-terminal tyrosine by carboxypeptidase P(CPP) in bold. Further C-terminal processing by dipeptidylcarboxypeptidase (DCP) was too slow to permit analysis of the subsequentdigestion steps (dotted arrows). FIG. 2B: Influence of dipeptidylcarboxypeptidase on C-terminal digestion. (SEQ ID NO:3) PQPQLPYPQPQLPYin phosphate buffered saline:Tris buffered saline=9:1 was digested byBBM alone or with addition of exogenous rabbit lung DCP (CortexBiochemicals, CA) or captopril. After overnight incubation, the fractionof accumulated SEQ ID NO:4) PQPQLPYPQPQLP (compared to initial amountsof (SEQ ID NO:3) PQPQLPYPQPQLPY at t=0 min) was analyzed as in FIG. 2A,but with an acetonitrile gradient of 20-65% in 6-35 minutes.

FIG. 3. Dose dependent acceleration of brush border mediated digestionby exogenous endoproteases. As seen from FIG. 2A-2B, the peptide (SEQ IDNO:4) PQPQLPYPQPQLP is stable toward further digestion. This peptide wasdigested with 27 ng/μl brush border membranes, either alone, withincreasing amounts of exogenous prolyl endopeptidase (PEP, specificactivity 28 μU/pg) from Flavobacterium meningosepticum (US Biological,MA), or with additional elastase (E-1250, Sigma, Mo.), bromelain(B-5144, Sigma, Mo.) or papain (P-5306, Sigma, Mo.) (12). After onehour, the fraction of remaining (SEQ ID NO:4) PQPQLPYPQPQLP (compared tothe initial amount at t=0 min) was analyzed and quantified as in FIG. 1.

FIG. 4. Products of gastric and pancreatic protease mediated digestionof α2-gliadin under physiological conditions. Analysis was performed byLC-MS. The longest peptides are highlighted by arrows and also in thesequence of α2-gliadin (inset).

FIG. 5. In vivo brush border membrane digestion of peptides. LC-UV₂₁₅traces of 25 μM of (SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPFbefore perfusion and after perfusion (residence time=20 min). LC-UV₂₁₅traces of 50 μM of SEQ ID NO:1 QLQPFPQPQLPY before perfusion and afterperfusion (residence time=20 min).

FIG. 6. Alignment of representative gluten and non-gluten peptideshomologous to (SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF.

FIG. 7. Breakdown and detoxification of 33-mer gliadin peptide with PEP.In vitro incubation of PEP (540 mU/ml) with the 33-mer gliadin peptide(100 μM) for the indicated time. In vivo digestion of the 33-mer gliadinpeptide (25 μM) with PEP (25 mU/ml) and the rat's intestine (residencetime=20 min).

FIG. 8. Effect of pH on the turnover numbers (_(kca)t) of FM PEP, MX PEPand SC PEP.

FIG. 9. Resistance of the FM PEP and the MX PEP to inactivation bygastric and pancreatic enzymes. Pancreatic enzyme stability wasevaluated by treating 5 U/ml of the FM PEP and the MX PEP with 1 mg/mltrypsin, 1 mg/ml chymotrypsin, 0.2 mg/ml elastase and 0.2 mg/mlcarboxypeptidase A (40 mM phosphate, pH=6.5). Pepsin stability wastested by treating the FM PEP and the MX PEP (5 U/ml) with 1 mg/mlpepsin (pH=2, 20 mM HCl).

FIG. 10. Site specificity of PQPQLPYPQPQLP hydrolysis by individualPEPs. HPLC-UV (215 nm) traces are shown for each reaction mixture.Initial cleavage fragments (100 μM (SEQ ID NO:4) PQPQLPYPQPQLP, 0.1 μMenzyme, t=5 min) were identified by tandem mass spectrometry. Thestarting material (SEQ ID NO:4) PQPQLPYPQPQLP and the cleavage fragmentsA: (SEQ ID NO:4, aa. 1-8) PQPQLPYP, B: (SEQ ID NO:4, aa 7-13) YPQPQLP,C: (SEQ ID NO:12, aa 1-6) PQPQLP, D: (SEQ ID NO:4, aa 2-6) QPQLP) areindicated in the traces.

FIGS. 11A-11C. Hydrolysis of (SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF by FM PEP, MX PEP and SC PEP. (A) Time dependence ofhydrolysis in the presence of 10 μM substrate and 0.1 μM enzyme. Thesubstrate appears as a doublet at a retention time of ca. 18 min, due tothe presence of equal quantities of the 32-mer from which the N-terminalLeu is deleted; presence of this contaminant does not affect analysis.From the residual peak areas, the rates of substrate (33-mer+32-mer)disappearance were calculated as 2.3 μM/min (FM PEP), 0.43 μM/min (MXPEP) and 0.07 μM/min (SC PEP). (B) Initial cleavage fragments observeddue to hydrolysis by FM PEP (t=1 min) and MX PEP (t=5 min). (C) Summaryof initial cleavage fragments from FM PEP and MX PEP catalyzedhydrolysis of the 33-mer substrate.

FIGS. 12A-12C. Competitive proteolysis of (SEQ ID NO:4) PQPQLPYPQPQLPand (SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF by each PEP. 10 μMof the longer peptide and 50 μM of the shorter peptide were co-incubatedwith 0.1 μM of (A) FM PEP; (B) MX PEP; (C) SC PEP.

FIGS. 13A-13B. Competitive proteolysis of (SEQ ID NO:4) PQPQLPYPQPQLP(50 μM) and (SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (10 μM) inthe presence of 30 mg/ml pepsin-treated gluten. This complex mixture ofsubstrates was treated under physiological conditions with a mixture ofpancreatic enzymes (trypsin, chymotrypsin, carboxypeptidase, elastase),brush border membrane enzymes (derived from rat small intestine) andeither (A) FM PEP or (B) MX PEP.

FIG. 14. (SEQ ID NO:12) Proteolysis of LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (5 μM) co-perfused with individual PEP's (0.1 μM) in the smallintestinal lumen of an anesthetized rat. Each enzyme-substrate mixturewas introduced via a catheter into a 15-20 cm segment of the upperjejunum. Samples were collected at the other end of the segment, andanalyzed by UV-HPLC (215 nm). The control without any PEP is shown inthe top trace.

FIGS. 15A and 15B. Analysis of rat intestinal content for gluten derivedpeptides in the absence or presence of enteric coated PEP capsules.

FIG. 16. Urine xyloses for patients with normal pre-stage xyloseabsorption and a positive xylose gluten challenge.

FIG. 17. Fecal fats for patients with normal to mild pre-stagesteatorrhea and a positive fecal fat gluten challenge.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Celiac Sprue and/or dermatitis herpetiformis are treated by digestion ofgluten oligopeptides contained in foodstuffs consumed by individualssuffering from one or both conditions. Gluten oligopeptides are highlyresistant to cleavage by gastric and pancreatic peptidases such aspepsin, trypsin, chymotrypsin, and the like. By providing for digestionof gluten oligopeptides with glutenase, oligopeptides are cleaved intofragments, thereby preventing the disease-causing toxicity.

Methods and compositions are provided for the administration of one ormore glutenases inhibitors to a patient suffering from Celiac Sprueand/or dermatitis herpetiformis. In some patients, these methods andcompositions will allow the patient to ingest glutens without serioushealth consequences, much the same as individuals that do not sufferfrom either of these conditions. In some embodiments, the formulationsof the invention comprise a glutenase contained in an enteric coatingthat allows delivery of the active agent(s) to the intestine; in otherembodiments, the active agent(s) is stabilized to resist digestion inacidic stomach conditions. In some cases the active agent(s) havehydrolytic activity under acidic pH conditions, and can thereforeinitiate the proteolytic process on toxic gluten sequences in thestomach itself. Alternative methods of administration provided by theinvention include genetic modification of patient cells, e.g.enterocytes, to express increased levels of glutenases; and theintroduction of micro-organisms expressing such glutenases so as totransiently or permanently colonize the patient's intestinal tract. Suchmodified patient cells (which include cells that are not derived fromthe patient but that are not immunologically rejected when administeredto the patient) and microorganisms of the invention are, in someembodiments, formulated in a pharmaceutically acceptable excipient, orintroduced in foods. In another embodiment, the invention provides foodspretreated or combined with a glutenase and methods for treating foodsto remove the toxic oligopeptides of gluten.

The methods of the invention can be used for prophylactic as well astherapeutic purposes. As used herein, the term “treating” refers both tothe prevention of disease and the treatment of a disease or apre-existing condition. The invention provides a significant advance inthe treatment of ongoing disease, to stabilize or improve the clinicalsymptoms of the patient. Such treatment is desirably performed prior toloss of function in the affected tissues but can also help to restorelost function or prevent further loss of function. Evidence oftherapeutic effect may be any diminution in the severity of disease,particularly as measured by the severity of symptoms such as fatigue,chronic diarrhea, malabsorption of nutrients, weight loss, abdominaldistension, anemia, and other symptoms of Celiac Sprue. Other diseaseindicia include the presence of antibodies specific for glutens, thepresence of antibodies specific for tissue transglutaminase, thepresence of pro-inflammatory T cells and cytokines, damage to the villusstructure of the small intestine as evidenced by histological or otherexamination, enhanced intestinal permeability, and the like.

Patients that may be treated by the methods of the invention includethose diagnosed with celiac sprue through one or more of serologicaltests, e.g. anti-gliadin antibodies, anti-transglutaminase antibodies,anti-endomysial antibodies; endoscopic evaluation, e.g. to identifyceliac lesions; histological assessment of small intestinal mucosa, e.g.to detect villous atrophy, crypt hyperplasia, infiltration ofintra-epithelial lymphocytes; and any GI symptoms dependent on inclusionof gluten in the diet. Amelioration of the above symptoms uponintroduction of a strict gluten-free diet is a key hallmark of thedisease. However, analysis of celiac patients has shown that a highlevel of patients believed to be in remission are, in fact, sufferingmalabsorption, as evidenced by indicia including, without limitation,xylose absorption tests, fecal fat analysis, lactulose/mannitolpermeability tests, and the like. In some embodiments of the invention,patients are evaluated by examination of intestinal malabsorption forinitial diagnosis, assessment, and/or monitoring during and aftertreatment.

These diagnostic approaches may be used in identifying patients who areresponsive to protease therapy, and for clinical evaluation of candidatetherapies. In particular, patient status may be assessed via ashort-term (2-4 week), low-dose (2-10 g/day) gluten challenge, whereintestinal malabsorption and malfunction (as judged by reduced xyloseabsorption, increased fecal fat excretion, and/or increasedlactulose/mannitol permeability) is expected to worsen in response tosuch a gluten challenge. Therapeutic efficacy of a clinical protocoland/or formulation can be assessed in a crossover mode, where thepatient is challenged alternately with gluten-only or withgluten+enzyme, separated by a washout period of 4-8 weeks, incombination with assessment of intestinal malabsorption. Alternatively,therapeutic efficacy can also be assessed by endoscopic evaluationduring which biopsy samples are collected from the duodenum and upperjejunum, and subjected to histological analysis.

Given the safety of oral proteases, they also find a prophylactic use inhigh-risk populations, such as Type I diabetics, family members ofdiagnosed celiac patients, HLA-DQ2 positive individuals, and/or patientswith gluten-associated symptoms that have not yet undergone formaldiagnosis. Such patients may be treated with regular-dose or low-dose(10-50% of the regular dose) enzyme. Similarly, temporary high-dose useof such an agent is also anticipated for patients recovering fromgluten-mediated enteropathy in whom gut function has not yet returned tonormal, for example as judged by fecal fat excretion assays.

Patients that can benefit from the present invention may be of any ageand include adults and children. Children in particular benefit fromprophylactic treatment, as prevention of early exposure to toxic glutenpeptides can prevent initial development of the disease. Childrensuitable for prophylaxis can be identified by genetic testing forpredisposition, e.g. by HLA typing; by family history, by T cell assay,or by other medical means. As is known in the art, dosages may beadjusted for pediatric use.

Although the present invention is not to be bound by any theory ofaction, it is believed that the primary event in Celiac Sprue requirescertain gluten oligopeptides to access antigen binding sites within thelamina propria region interior to the relatively impermeable surfaceintestinal epithelial layer. Ordinarily, oligopeptide end products ofpancreatic protease processing are rapidly and efficiently hydrolyzedinto amino acids and/or di- or tri-peptides by intestinal peptidasesbefore they are transported across the epithelial layer. The enzymes ofthe GI tract proteolyze gluten slowly due to the proline- andglutamine-rich character of this important dietary protein source.Furthermore, the Celiac Sprue toxicity of gluten resides in the proline-and glutamine-rich segments of gluten. Therefore proteases withspecificity toward proline and glutamine residues are expected to beuseful for treating Celiac Sprue.

The normal assimilation of dietary proteins by the human gut can bedivided into three major phases: (i) initiation of proteolysis in thestomach by pepsin and highly efficient endo- and C-terminal cleavage inthe upper small intestine cavity (duodenum) by secreted pancreaticproteases and carboxypeptidases; (ii) further processing of theresulting oligopeptide fragments by exo- and endopeptidases anchored inthe brush border surface membrane of the upper small intestinalepithelium (jejunum); and (iii) facilitated transport of the resultingamino acids, di- and tripeptides across the epithelial cells into thelamina propria, from where these nutrients enter capillaries fordistribution throughout the body.

Because most proteases and peptidases normally present in the humanstomach and small intestine are unable to hydrolyze the amide bonds ofproline residues, it is shown herein that the abundance of proline andglutamine residues in gliadins and related proteins from wheat, rye andbarley can constitute a major digestive obstacle for the enzymesinvolved in phases (i) and (ii) above. This leads to an increasedconcentration of relatively stable gluten derived oligopeptides in thegut. Furthermore, because aminopeptidase and especially carboxypeptidaseactivity towards oligopeptides with proline residues at the N- andC-termini, respectively, is low in the small intestine, detoxificationof gluten oligopeptides in phase (iii) above is also slow. Byadministering peptidases capable of cleaving such gluten oligopeptidesin accordance with the methods of the invention, the amount of toxicpeptides is diminished, thereby slowing or blocking disease progression.

Tissue transglutaminase (tTGase), an enzyme found on the extracellularsurface in many organs including the intestine, catalyzes the formationof isopeptide bonds between glutamine and lysine residues of differentpolypeptides, leading to protein-protein crosslinks in the extracellularmatrix. The enzyme tTGase is the primary focus of the autoantibodyresponse in Celiac Sprue. Gliadins, secalins and hordeins containseveral sequences rich in Pro-Gln residues that are high-affinitysubstrates for tTGase; tTGase catalyzed deamidation of at least some ofthese sequences dramatically increases their affinity for HLA-DQ2, theclass II MHC allele present in >90% Celiac Sprue patients. Presentationof these deamidated epitopes by DQ2 positive antigen presenting cellseffectively stimulates proliferation of gliadin-specific T cells fromintestinal biopsies of most Celiac Sprue patients. The toxic effects ofgluten include immunogenicity of the gluten oligopeptides, leading toinflammation; the lectin theory predicts that gliadin peptides may alsodirectly bind to surface receptors.

The present invention relates generally to methods and reagents usefulin treating foodstuffs containing gluten with enzymes that digest theoligopeptides toxic to Celiac Sprue patients. Although specific enzymesare exemplified herein, any of a number of alternative enzymes andmethods apparent to those of skill in the art upon contemplation of thisdisclosure are equally applicable and suitable for use in practicing theinvention. The methods of the invention, as well as tests to determinetheir efficacy in a particular patient or application, can be carriedout in accordance with the teachings herein using procedures standard inthe art. Thus, the practice of the present invention may employconventional techniques of molecular biology (including recombinanttechniques), microbiology, cell biology, biochemistry and immunologywithin the scope of those of skill in the art. Such techniques areexplained fully in the literature, such as, “Molecular Cloning: ALaboratory Manual”, second edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Handbook of Experimental Immunology” (D. M. Weir & C. C.Blackwell, eds.); “Gene Transfer Vectors for Mammalian Cells” (J. M.Miller & M. P. Calos, eds., 1987); “Current Protocols in MolecularBiology” (F. M. Ausubel et al., eds., 1987); “PCR: The Polymerase ChainReaction” (Mullis et al., eds., 1994); and “Current Protocols inImmunology” (J. E. Coligan et al., eds., 1991); as well as updated orrevised editions of all of the foregoing.

As used herein, the term “glutenase” refers to an enzyme useful in themethods of the present invention that is capable, alone or incombination with endogenous or exogenously added enzymes, of cleavingtoxic oligopeptides of gluten proteins of wheat, barley, oats and ryeinto non-toxic fragments. Gluten is the protein fraction in cerealdough, which can be subdivided into glutenins and prolamines, which aresubclassified as gliadins, secalins, hordeins, and avenins from wheat,rye, barley and oat, respectively. For further discussion of glutenproteins, see the review by Wieser (1996) Acta Paediatr Suppl. 412:3-9,incorporated herein by reference.

In one embodiment, the term “glutenase” as used herein refers to aprotease or a peptidase enzyme that meets one or more of the criteriaprovided herein. Using these criteria, one of skill in the art candetermine the suitability of a candidate enzyme for use in the methodsof the invention. Many enzymes will meet multiple criteria, includingtwo, three, four or more of the criteria, and some enzymes will meet allof the criteria. The terms “protease” or “peptidase” can refer to aglutenase and as used herein describe a protein or fragment thereof withthe capability of cleaving peptide bonds, where the scissile peptidebond may either be terminal or internal in oligopeptides or largerproteins. Prolyl-specific peptidases are glutenases useful in thepractice of the present invention.

Glutenases of the invention include protease and peptidase enzymeshaving at least about 20% sequence identity at the amino acid level,more usually at least about 40% sequence identity, and preferably atleast about 70% sequence identity to one of the following peptidases:prolyl endopeptidase (PEP) from F. meningosepticum (Genbank accessionnumber D10980), PEP from A. hydrophila (Genbank accession numberD14005), PEP form S. capsulata (Genbank accession number AB010298), DCPI from rabbit (Genbank accession number X62551), DPP IV from Aspergillusfumigatus (Genbank accession number U87950) or cysteine proteinase Bfrom Hordeum vulgare (Genbank accession number JQ1110).

Each of the above proteases described herein can be engineered toimprove desired properties such as enhanced specificity toward toxicgliadin sequences, improved tolerance for longer substrates, acidstability, pepsin resistance, resistance to proteolysis by thepancreatic enzymes and improved shelf-life. The desired property can beengineered via standard protein engineering methods.

In one embodiment of the present invention, the glutenase is a PEP.Homology-based identification (for example, by a PILEUP sequenceanalysis) of prolyl endopeptidases can be routinely performed by thoseof skill in the art upon contemplation of this disclosure to identifyPEPs suitable for use in the methods of the present invention. PEPs areproduced in microorganisms, plants and animals. PEPs belong to theserine protease superfamily of enzymes and have a conserved catalytictriad composed of a Ser, His, and Asp residues. Some of these homologshave been characterized, e.g. the enzymes from F. meningoscepticum,Aeromonas hydrophila, Aeromonas punctata, Novosphingobium capsulatum,Pyrococcus furiosus and from mammalian sources are biochemicallycharacterized PEPs. Others such as the Nostoc and Arabidopsis enzymesare likely to be PEPs but have not been fully characterized to date.Homologs of the enzymes of interest may be found in publicly availablesequence databases, and the methods of the invention include suchhomologs. Candidate enzymes are expressed using standard heterologousexpression technologies, and their properties are evaluated using theassays described herein.

In one embodiment of the invention, the glutenase is Flavobacteriummeningosepticum PEP (Genbank ID # D10980). Relative to the F.meningoscepticum enzyme, the pairwise sequence identity of this familyof enzymes is in the 30-60% range. Accordingly, PEPs include enzymeshaving >30% identity to the F. meningoscepticum enzyme (as in thePyrococcus enzymes), or having >40% identity (as in the Novosphingobiumenzymes), or having >50% identity (as in the Aeromonas enzymes) to theF. meningoscepticum enzyme. A variety of assays have verified thetherapeutic utility of this PEP. In vitro, this enzyme has been shown torapidly cleave several toxic gluten peptides, including the highlyinflammatory 33-mer, (SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF.In vivo it acts synergistically with the peptidases of the intestinalbrush border membrane so as to rapidly detoxify these peptides, as wellas gluten that has been pre-treated with gastric and pancreaticproteases. It has broad chain length specificity, making it especiallywell suited for the breakdown of long proline-rich peptides releasedinto the duodenum from the stomach. The enzyme has a pH optimum aroundpH 7, and has high specific activity under conditions that mimic theweakly acidic environment of the upper small intestine. FlavobacteriumPEP can cleave all T cell epitopes in gluten that have been tested todate. It has particular preference for the immunodominant epitopes foundin alpha-gliadin. When grocery-store gluten is treated with this PEP, arapid decrease in its antigenicity can be observed, as judged by LC-MSanalysis and testing against polyclonal T cell lines derived from smallintestinal biopsies from Celiac Sprue patients. The denatured protein isnon-allergenic in rodents, rabbits and humans. It is relatively stabletoward destruction by pancreatic proteases, an important feature sinceunder physiological conditions it will be expected to act in concertwith those enzymes.

Another enzyme of interest is Myxococcus xanthus PEP (Genbank ID#AF127082) (SEQ ID NO:47). This enzyme possesses many of the advantagesof the Flavobacterium PEP. It can cleave the 33-mer into small non-toxicpeptides. Whereas the Flavobacterium enzyme appears to have a relativelystrict preference for PQ bonds in gliadin peptides, the Myxococcusenzyme can cleave at PQ, PY and PF bonds, a feature that allows it toproteolyze a broader range of gluten epitopes. Compared to theFlavobacterium enzyme, it has equivalent stability toward the pancreaticproteases and superior stability toward acidic environments. TheMyxococcus enzyme is well expressed in E. coli, making it feasible toproduce this enzyme cheaply.

Another enzyme of interest is Sphingomonas capsulata PEP (Genbank ID#AB010298). This enzyme is comparable to the Flavobacterium andMyxococcus enzyme. It has broader sequence specificity than either theFlavobacterium or the Myxococcus PEP, and may therefore be able todestroy the widest range of antigenic epitopes. Like the Myxococcusenzyme, it is also well expressed in E. coli.

Another enzyme of interest is Lactobacillus helveticus PEP (GenbankID#321529). Unlike the above PEPs, this PEP is a zinc enzyme. It canefficiently proteolyze long peptide substrates such as the caseinpeptides (SEQ ID NO:28) YQEPVLGPVRGPFPIIV and (SEQ ID NO:29) RPKHPIKHQ.Proteolysis occurs at all PV and PI subsites, suggesting the PEP prefershydrophobic residues at the 51′ position, as are frequently found ingluten. Since the producer strain of L. helveticus CNRZ32 is commonlyused in cheesemaking, this enzyme has desirable properties as afood-grade enzyme.

Another enzyme of interest is Penicillium citrinum PEP (Genbank ID#D25535). This enzyme has been shown to possess PEP activity based on itsability to effectively cleave a number of Pro-Xaa bonds in peptides suchas dynorphin A and substance P. The putative metalloprotease has theadvantages of small size and a pH profile that renders it suitable toworking in concert with the pancreatic enzymes in the duodenum. As such,it is a good candidate for the treatment of Celiac Sprue.

Other than proline, glutamine residues are also highly prevalent ingluten proteins. The toxicity of gluten in Celiac Sprue has beendirectly correlated to the presence of specific Gln residues. Therefore,glutamine-specific proteases are also beneficial for the treatment ofCeliac Sprue. Since oats contain proteins that are rich in glutamine butnot especially rich in proline residues, an additional benefit of aglutamine-specific protease is the improvement of oat tolerance in thoseceliac patients who show mild oat-intolerance. An example of such aprotease is the above-mentioned cysteine endoproteinase that cleavesgluten proteins rapidly with a distinct preference for post-Glncleavage. This enzyme is Hordeum vulgare endoprotease (Genbank accessionU19384), which has been shown to efficiently digest .alpha.2-gliadin.The enzyme is active under acidic conditions, and is useful as an orallyadministered dietary supplement. A gluten-containing diet may besupplemented with orally administered proEPB2, resulting in effectivedegradation of immunogenic gluten peptides in the acidic stomach, beforethese peptides enter the intestine and are presented to the immunesystem. Proteins with high sequence similarity to this enzyme are alsoof interest. An advantage of these enzymes is that they are consideredas safe for human oral consumption, due to their presence in dietarygluten from barley.

Intestinal dipeptidyl peptidase IV and dipeptidyl carboxypeptidase I arethe rate-limiting enzymes in the breakdown of toxic gliadin peptidesfrom gluten. These enzymes are bottlenecks in gluten digestion in themammalian small intestine because (i) their specific activity isrelatively low compared to other amino- and carboxy-peptidases in theintestinal brush border; and (ii) due to their strong sensitivity tosubstrate chain length, they cleave long immunotoxic peptides such asthe 33-mer extremely slowly. Both these problems can be amelioratedthrough the administration of proline-specific amino- andcarboxy-peptidases from other sources. For example the X-Pro dipeptidasefrom Aspergillus oryzae (GenBank ID# BD191984) and the carboxypeptidasefrom Aspergillus saitoi (GenBank ID# D25288) can improve glutendigestion in the Celiac intestine.

A glutenase of the invention includes a peptidase or protease that has aspecific activity of at least 2.5 U/mg, preferably 25 U/mg and morepreferably 250 U/mg for cleavage of a peptide comprising one of more ofthe following motifs: Gly-Pro-pNA, Z-Gly-Pro-pNA (where Z is abenzyloxycarbonyl group), and Hip-His-Leu, where “Hip” is hippuric acid,pNA is para-nitroanilide, and 1 U is the amount of enzyme required tocatalyze the turnover of 1 μmole of substrate per minute. Chromogenicsubstrates may be utilized in screening, e.g. substrates such asCbz-Gly-Pro-pNA or Suc-Ala-Pro-pNA enables identification ofproline-specific proteases. Similar substrates can also be used toidentify glutamine-specific proteases. These assays can be monitored byUV-Vis spectrophotometric methods.

A glutenase of the invention includes an enzyme belonging to any of thefollowing enzyme classifications: EC 3.4.21.26, EC 3.4.14.5, or EC3.4.15.1.

A glutenase of the invention includes an enzyme having a kcat/Km of atleast about 2.5 s⁻¹ M⁻¹, usually at least about 250 s⁻¹ M⁻¹ andpreferably at least about 25000 s⁻¹ M⁻¹ for cleavage of any of thefollowing peptides, including known T cell epitopes in gluten, underoptimal conditions: (SEQ ID NO:1) QLQPFPQPQLPY or PFPQPQLPY, (SEQ IDNO:3) PQPQLPYPQPQLPY or PQPQLPYPQ, (SEQ ID NO:13) QPQQSFPQQQ orPQQSFPQQQ, (SEQ ID NO:14) QLQPFPQPELPY, (SEQ ID NO:15) PQPELPYPQPELPY,(SEQ ID NO:16) QPQQSFPEQQ; (SEQ ID NO: 30) IQPQQPAQL; (SEQ ID NO:31)QQPQQPYPQ; (SEQ ID NO:32) SQPQQQFPQ; (SEQ ID NO:33) QQPFPQQPQ; or (SEQID NO:34) PFSQQQQPV. Cleavage of longer, physiologically generatedpeptides containing one or more of the above epitopes may also beassessed, for example cleavage of the 33-mer from alpha-gliadin, (SEQ IDNO:12) LQLQPF(PQPQLPY)₃PQPQPF, and the 26-mer from gamma-gliadin, (SEQID NO:35) FLQPQQPFPQQPQQPYPQQPQQPFPQ. A glutenase of the inventionincludes peptidase or protease having a specificity kcat/Km >2 mM⁻¹ s⁻¹for the quenched fluorogenic substrate (SEQ ID NO:36)Abz-QPQQP-Tyr(NO₂)-D. These assays can be monitored by HPLC orfluorescence spectroscopy. For the latter assays, suitable fluorophorescan be attached to the amino- and carboxy-termini of the peptides.

A glutenase useful in the practice of the present invention can beidentified by its ability to cleave a pretreated substrate to removetoxic gluten oligopeptides, where a “pretreated substrate” is a gliadin,hordein, secalin or avenin protein that has been treated withphysiological quantities of gastric and pancreatic proteases, includingpepsin (1:100 mass ratio), trypsin (1:100), chymotrypsin (1:100),elastase (1:500), and carboxypeptidases A and B (1:100). Pepsindigestion may be performed at pH 2 for 20 min., to mimic gastricdigestion, followed by further treatment of the reaction mixture withtrypsin, chymotrypsin, elastase and carboxypeptidase at pH 7 for 1 hour,to mimic duodenal digestion by secreted pancreatic enzymes. Thepretreated substrate comprises oligopeptides resistant to digestion,e.g. under physiological conditions. A glutenase may catalyze cleavageof pepsin-trypsin-chymotrypsin-elastase-carboxypeptidase (PTCEC) treatedgluten such that less than 10% of the products are longer than (SEQ IDNO:3, aa 1-9) PQPQLPYPQ (as judged by longer retention times on a C18reverse phase HPLC column monitored at A₂₁₅).

The ability of a peptidase or protease to cleave a pretreated substratecan be determined by measuring the ability of an enzyme to increase theconcentration of free NH₂-termini in a reaction mixture containing 1mg/ml pretreated substrate and 10 μg/ml of the peptidase or protease,incubated at 37° C. for 1 hour. A glutenase useful in the practice ofthe present invention will increase the concentration of the free aminotermini under such conditions, usually by at least about 25%, moreusually by at least about 50%, and preferably by at least about 100%. Aglutenase includes an enzyme capable of reducing the residual molarconcentration of oligopeptides greater than about 1000 Da in a 1 mg/ml“pretreated substrate” after a 1 hour incubation with 10 μg/ml of theenzyme by at least about 2-fold, usually by at least about 5-fold, andpreferably by at least about 10-fold. The concentration of sucholigopeptides can be estimated by methods known in the art, for examplesize exclusion chromatography and the like.

A glutenase of the invention includes an enzyme capable ofdetoxification of whole gluten, as monitored by polyclonal T cell linesderived from intestinal biopsies of celiac patients; detoxification ofwhole gluten as monitored by LC-MS-MS; and/or detoxification of wholegluten as monitored by ELISA assays using monoclonal antibodies capableof recognizing sequences specific to gliadin.

For example, a glutenase may reduce the potency by which a “pretreatedsubstrate” can antagonize binding of (SEQ ID NO:17) PQPELPYPQPQLP toHLA-DQ2. The ability of a substrate to bind to HLA-DQ is indicative ofits toxicity; fragments smaller than about 8 amino acids are generallynot stably bound to Class II MHC. Treatment with a glutenase thatdigests toxic oligopeptides, by reducing the concentration of the toxicoligopeptides, prevents a mixture containing them from competing with atest peptide for MHC binding. To test whether a candidate glutenase canbe used for purposes of the present invention, a 1 mg/ml solution of“pretreated substrate” may be first incubated with 10 μg/ml of thecandidate glutenase, and the ability of the resulting solution todisplace radioactive (SEQ ID NO:18) PQPELPYPQPQPLP pre-bound to HLA-DQ2molecules can then be quantified, with a reduction of displacement,relative to a non-treated control, indicative of utility in the methodsof the present invention.

A glutenase of the invention includes an enzyme that reduces theanti-tTG antibody response to a “gluten challenge diet” in a CeliacSprue patient by at least about 2-fold, more usually by at least about5-fold, and preferably by at least about 10-fold. A “gluten challengediet” is defined as the intake of 100 g bread per day for 3 days by anadult Celiac Sprue patient previously on a gluten-free diet. Theanti-tTG antibody response can be measured in peripheral blood usingstandard clinical diagnostic procedures, as known in the art.

Excluded from the term “glutenase” are the following peptidases: humanpepsin, human trypsin, human chymotrypsin, human elastase, papayapapain, and pineapple bromelain, and usually excluded are enzymes havinggreater than 98% sequence identity at the amino acid level to suchpeptidases, more usually excluded are enzymes having greater than 90%sequence identity at the amino acid level to such peptidases, andpreferably excluded are enzymes having greater than 70% sequenceidentity at the amino acid level to such peptidases.

Among gluten proteins with potential harmful effect to Celiac Spruepatients are included the storage proteins of wheat, species of whichinclude Triticum aestivum; Triticum aethiopicum; Triticum baeoticum;Triticum militinae; Triticum monococcum; Triticum sinskajae; Triticumtimopheevii; Triticum turgidum; Triticum urartu, Triticum vavilovii;Triticum zhukovskyi; etc. A review of the genes encoding wheat storageproteins may be found in Colot (1990) Genet Eng (N Y) 12:225-41. Gliadinis the alcohol-soluble protein fraction of wheat gluten. Gliadins aretypically rich in glutamine and proline, particularly in the N-terminalpart. For example, the first 100 amino acids of α- and γ-gliadinscontain ˜35% and ˜20% of glutamine and proline residues, respectively.Many wheat gliadins have been characterized, and as there are manystrains of wheat and other cereals, it is anticipated that many moresequences will be identified using routine methods of molecular biology.In one aspect of the present invention, genetically modified plants areprovided that differ from their naturally occurring counterparts byhaving gliadin proteins that contain a reduced content of glutamine andproline residues.

Examples of gliadin sequences include but are not limited to wheat alphagliadin sequences, for example as provided in Genbank, accession numbersAJ133612; AJ133611; AJ133610; AJ133609; AJ133608; AJ133607; AJ133606;AJ133605; AJ133604; AJ133603; AJ133602; D84341.1; U51307; U51306;U51304; U51303; U50984; and U08287. A sequence of wheat omega gliadin isset forth in Genbank accession number AF280605.

For the purposes of the present invention, toxic gliadin oligopeptidesare peptides derived during normal human digestion of gliadins andrelated storage proteins as described above, from dietary cereals, e.g.wheat, rye, barley, and the like. Such oligopeptides are believed to actas antigens for T cells in Celiac Sprue. For binding to Class II MHCproteins, immunogenic peptides are usually from about 8 to 20 aminoacids in length, more usually from about 10 to 18 amino acids. Suchpeptides may include PXP motifs, such as the motif PQPQLP (SEQ ID NO:8).Determination of whether an oligopeptide is immunogenic for a particularpatient is readily determined by standard T cell activation and otherassays known to those of skill in the art.

As demonstrated herein, during digestion, peptidase resistantoligopeptides remain after exposure of glutens, e.g. gliadin, to normaldigestive enzymes. Examples of peptidase resistant oligopeptides areprovided, for example, as set forth in SEQ ID NO:5, 6, 7 and 10. Otherexamples of immunogenic gliadin oligopeptides are described in Wieser(1995) Baillieres Clin Gastroenterol 9(2):191-207, incorporated hereinby reference.

Determination of whether a candidate enzyme will digest a toxic glutenoligopeptide, as discussed above, can be empirically determined. Forexample, a candidate may be combined with an oligopeptide comprising oneor more Gly-Pro-pNA, Z-Gly-Pro-pNA, Hip-His-Leu, Abz-QLP-Tyr(NO₂)-PQ,Abz-PYPQPQ-Tyr(NO₂), PQP-Lys(Abz)-LP-Tyr(NO₂)—PQPQLP,PQPQLP-Tyr(NO₂)—PQP-Lys(Abz)-LP motifs; with one or more of theoligopeptides (SEQ ID NO:1) QLQPFPQPQLPY, (SEQ ID NO:3) PQPQLPYPQPQLPY,(SEQ ID NO:13) QPQQSFPQQQ, (SEQ ID NO:14) QLQPFPQPELPY, (SEQ ID NO:15)PQPELPYPQPELPY, (SEQ ID NO:16) QPQQSFPEQQ or (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF; or with a pretreated substratecomprising one or more of gliadin, hordein, secalin or avenin proteinsthat have been treated with physiological quantities of gastric andpancreatic proteases. In each instance, the candidate is determined tobe a glutenase of the invention if it is capable of cleaving theoligopeptide. Glutenases that have a low toxicity for human cells andare active in the physiologic conditions present in the intestinal brushborder are preferred for use in some applications of the invention, andtherefore it may be useful to screen for such properties in candidateglutenases.

Measurements of gastrointestinal tract (GIT) permeability andmalabsorption are useful in the analysis patients for therapy, ofmonitoring therapy, and for assessment of candidate agents, formulationsand dosing protocols. Detoxification of whole gluten may be judged by adouble-blinded crossover trial in celiac patients administered with 5-20g gluten/day for 2 weeks. Gluten induced intestinal malfunction can bemeasured by standard absorption tests before and after each two weekperiod. Intestinal malabsorption and malfunction is expected to worsenin response to such a gluten challenge, unless the gluten is detoxifiedby the glutenase or combination of glutenases.

Various such tests are known in the art. Tests of interest includeintestinal permeability measured by a human alpha-lactalbumin,beta-lactoglobulin, mannitol, and/or lactulose test, or gastricpermeability with a sucrose test (see Vogelsang et al. (1996)Gastroenterology 111:1, 73-7; Kuitunen and Savilahti (1996) J PediatrGastroenterol Nutr. 22:2, 197-204). The absorption of lactulose andmannitol may also be measured in serum after oral ingestion of testsugars, e.g. by HPLC determination of lactulose and mannitol (seeFleming et al. (1996) Clin Chem. 42:3, 445-8).

D-xylose is a pentose sugar which when ingested, is absorbed from thejejunum and excreted, largely unchanged, in the urine. Absorption ofxylose is a measure of the intestine's ability to absorbmonosaccharides. No standard protocol is defined. A 25 g bolus is widelyused being taken with 500 ml of water after an overnight fast.Absorption is assessed from urine specimens collected over a 5 hourperiod or alternatively, from blood xylose measured 1-2 hours afteringestion. Normal results are serum xylose concentration at 1 hr inexcess of 1.3 mM, urinary xylose excretion in excess of 4 g/5 hr.Hydrogen breath tests (H2-BT) are also used to diagnose carbohydratemalabsorption. Alveolar breath samples may be obtained beforeadministering orally 25 g of D-xylose and thereafter at 30 min intervalsfor 5 hr. Samples are analyzed for H2 by chromatography (see Casellas etal. (1996) Dig Dis Sci. 41:10, 2106-11).

Other tests monitor fat malabsorption. For example, in a 72-hour fecalfat challenge, 100 g fat is ingested for three days, and the fat instool measured for three days. In a normal patient the fecal fat will beless than about 7 g/day, while abnormal readings may be from about 10-60g/day. Quantitative tests may also find use, for example an oil red 0stain of fecal matter.

Schilling's test measures B12 absorption. Excess intake of radioactive(cobalt-57) B12 gets excreted in urine. The patient is loaded ahead ofthe test with IM B12, and then administered oral radioactive B12; whereurinary excretion is measured.

The oligopeptide or protein substrates for such assays may be preparedin accordance with conventional techniques, such as synthesis,recombinant techniques, isolation from natural sources, or the like. Forexample, solid-phase peptide synthesis involves the successive additionof amino acids to create a linear peptide chain (see Merrifield (1963)J. Am. Chem. Soc. 85:2149-2154). Recombinant DNA technology can also beused to produce the peptide.

Candidate glutenases for use in the practice of the present inventioncan be obtained from a wide variety of sources, including libraries ofnatural and synthetic proteins. For example, numerous means areavailable for random and directed mutation of proteins. Alternatively,libraries of natural proteins in the form of bacterial, fungal, plantand animal extracts are available or readily produced. Extracts ofgerminating wheat and other grasses is of interest as a source ofcandidate enzymes. Natural or synthetically produced libraries andcompounds are readily modified through conventional chemical, physicaland biochemical means, and such means can be used to producecombinatorial libraries. Known pharmacological agents may be subjectedto directed or random chemical modifications, such as acylation,alkylation, esterification, and amidification, to produce structuralanalogs of proteins.

Generally, a variety of assay mixtures are run in parallel withdifferent peptidase concentrations to obtain a differential response tothe various concentrations. Typically, one of these concentrationsserves as a negative control, i.e. at zero concentration or below thelevel of detection. A variety of other reagents may be included in ascreening assay. These include reagents like salts, detergents, and thelike that are used to facilitate optimal activity and/or reducenon-specific or background interactions. Reagents that improve theefficiency of the assay may be used. The mixture of components is addedin any order that provides for the requisite activity. Incubations areperformed at any suitable temperature, typically between 4 and 40° C.Incubation periods are selected for optimum activity but can also beoptimized to facilitate rapid high-throughput screening or otherpurposes. Typically, between 0.1 and 1 hours will be sufficient.

The level of digestion of the toxic oligopeptide can be compared to abaseline value. The disappearance of the starting material and/or thepresence of digestion products can be monitored by conventional methods.For example, a detectable marker can be conjugated to a peptide, and thechange in molecular weight associated with the marker is thendetermined, e.g. acid precipitation, molecular weight exclusion, and thelike. The baseline value can be a value for a control sample or astatistical value that is representative a control population. Variouscontrols can be conducted to ensure that an observed activity isauthentic, including running parallel reactions, positive and negativecontrols, dose response, and the like.

Active glutenases identified by the screening methods described hereincan serve as lead compounds for the synthesis of analog compounds toidentify glutenases with improved properties. Identification of analogcompounds can be performed through use of techniques such asself-consistent field (SCF) analysis, configuration interaction (CI)analysis, and normal mode dynamics analysis.

In one embodiment of the invention, the glutenase is a prolylendopeptidase (PEP, EC 3.4.21.26). Prolyl endopeptidases are widelydistributed in microorganisms, plants and animals, and have been clonedfrom Flavobacterium meningosepticum, (Yoshimoto et al. (1991) J.Biochem. 110, 873-8); Aeromonas hydrophyla (Kanatani et al. (1993) J.Biochem. 113, 790-6); Sphingomonas capsulata (Kabashima et al. (1998)Arch. Biochem. Biophys. 358, 141-148), Pyrococcus furious (Robinson etal. (1995) Gene 152, 103-6); pig (Rennex et al. (1991) Biochemistry 30,2195-2030); and the like. The suitability of a particular enzyme isreadily determined by the assays described above, by clinical testing,determination of stability in formulations, and the like. Other sourcesof PEP include Lactobacilli (Habibi-Najafi et al. (1994) J. Dairy Sci.77, 385-392), from where the gene of interest can be readily clonedbased on sequence homology to the above PEP's or via standard reversegenetic procedures involving purification, amino-acid sequencing,reverse translation, and cloning of the gene encoding the targetextracellular enzyme.

In another embodiment of the invention, glutenases are peptidasespresent in the brush border, which are supplemented. Formulations ofinterest may comprise such enzymes in combination with other peptidases.Peptidases present in brush border include dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5), and dipeptidyl carboxypeptidase (DCP, EC 3.4.15.1).The human form of these proteins may be used, or modified forms may beisolated from other suitable sources. Example of DPP IV enzymes includeAspergillus spp. (e.g. Byun et al. (2001) J. Agric. Food Chem. 49,2061-2063), ruminant bacteria such as Prevotella albensis M384 (NCBIprotein database Locus # CAC42932), dental bacteria such asPorphyromonas gingivalis W83 (Kumugai et al. (2000) Infect. Immun. 68,716-724), lactobacilli such as Lactobacillus helveticus (e.g. Vesanto,et al, (1995) Microbiol. 141, 3067-3075), and Lactococcus lactis (Mayoet al., (1991) Appl. Environ. Microbiol. 57, 38-44). Other DPP IVcandidates can readily be recognized based on homology to the aboveenzymes, preferably >30% sequence identity. Similarly, secreteddipeptidyl carboxypeptidases that cleave C-terminal X-Pro sequences arefound in many microbial sources including Pseudomonas spp (e.g.Ogasawara et al, (1997) Biosci. Biotechnol. Biochem. 61, 858-863),Streptomyces spp. (e.g. Miyoshi et al., (1992) J. Biochem. 112, 253-257)and Aspergilli spp. (e.g. Ichishima et al., (1977) J. Biochem. 81,1733-1737). Of particular interest is the enzyme from Aspergillus saitoi(Ichishima), due to its high activity at acidic pH values. Although thegenes encoding many of these enzymes have not yet been cloned, they canbe readily cloned by standard reverse genetic procedures. The DCP Ienzymes can be purified from the extracellular medium based on theirability to hydrolyze (SEQ ID NO:19) Z-Gly-Pro-Leu-Gly-Pro, Z-Gly-Pro, orHip-Gly-Pro. Alternatively, putative DCP I genes can be identified basedon homology to the E. coli enzyme (NCBI protein database LocusCAA41014.)

In another embodiment of the invention, glutenases are endoproteasesfound in developing grains of toxic cereals such as wheat, barley andrye. For example, Dominguez and Cejudo (Plant Physiol. 112, 1211-1217,1996) have shown that the endosperm of wheat (i.e. the part of the grainthat contains gliadin and glutenin) contains a variety of neutral andacid proteases. Although these proteases have not been individuallycharacterized, they are expected to be an especially rich source ofglutenases. Moreover, although the genes encoding these proteases havenot yet been cloned, Dominguez and Cejudo have established a convenientSDS-PAGE assay for identification and separation of these proteases.After excision of the corresponding protein bands from the gel, limitedsequence information can be obtained. The cDNA encoding these proteasescan therefore be readily cloned from this information using establishedreverse genetic procedures, and expressed in heterologous bacterial orfungal hosts. Of particular interest are proteases that hydrolyzeα2-gliadin within the 33-mer amino acid sequence identified in Example 2below. Of further interest are the subset of these proteases that retainactivity at acidic pH values (pH2-5) encountered in the stomach.

The amino acid sequence of a glutenase, e.g. a naturally occurringglutenase, can be altered in various ways known in the art to generatetargeted changes in sequence and additional glutenase enzymes useful inthe formulations and compositions of the invention. Such variants willtypically be functionally-preserved variants, which differ, usually insequence, from the corresponding native or parent protein but stillretain the desired biological activity. Variants also include fragmentsof a glutenase that retain enzymatic activity. Various methods known inthe art can be used to generate targeted changes, e.g. phage display incombination with random and targeted mutations, introduction of scanningmutations, and the like.

A variant can be substantially similar to a native sequence, i.e.differing by at least one amino acid, and can differ by at least two butusually not more than about ten amino acids (the number of differencesdepending on the size of the native sequence). The sequence changes maybe substitutions, insertions or deletions. Scanning mutations thatsystematically introduce alanine, or other residues, may be used todetermine key amino acids. Conservative amino acid substitutionstypically include substitutions within the following groups: (glycine,alanine); (valine, isoleucine, leucine); (aspartic acid, glutamic acid);(asparagine, glutamine); (serine, threonine); (lysine, arginine); and(phenylalanine, tyrosine).

Glutenase fragments of interest include fragments of at least about 20contiguous amino acids, more usually at least about 50 contiguous aminoacids, and may comprise 100 or more amino acids, up to the completeprotein, and may extend further to comprise additional sequences. Ineach case, the key criterion is whether the fragment retains the abilityto digest the toxic oligopeptides that contribute to the symptoms ofCeliac Sprue.

Modifications of interest that do not alter primary sequence includechemical derivatization of proteins, e.g., acetylation or carboxylation.Also included are modifications of glycosylation, e.g. those made bymodifying the glycosylation patterns of a protein during its synthesisand processing or in further processing steps; e.g. by exposing theprotein to enzymes that affect glycosylation, such as mammalianglycosylating or deglycosylating enzymes. Also embraced are sequencesthat have phosphorylated amino acid residues, e.g. phosphotyrosine,phosphoserine, or phosphothreonine.

Also useful in the practice of the present invention are proteins thathave been modified using molecular biological techniques and/orchemistry so as to improve their resistance to proteolytic degradationand/or to acidic conditions such as those found in the stomach, and tooptimize solubility properties or to render them more suitable as atherapeutic agent. For example, the backbone of the peptidase can becyclized to enhance stability (see Friedler et al. (2000) J. Biol. Chem.275:23783-23789). Analogs of such proteins include those containingresidues other than naturally occurring L-amino acids, e.g. D-aminoacids or non-naturally occurring synthetic amino acids.

The glutenase proteins of the present invention may be prepared by invitro synthesis, using conventional methods as known in the art. Variouscommercial synthetic apparatuses are available, for example, automatedsynthesizers by Applied Biosystems, Inc., Foster City, Calif., Beckman,and other manufacturers. Using synthesizers, one can readily substitutefor the naturally occurring amino acids one or more unnatural aminoacids. The particular sequence and the manner of preparation will bedetermined by convenience, economics, purity required, and the like. Ifdesired, various groups can be introduced into the protein duringsynthesis that allow for linking to other molecules or to a surface. Forexample, cysteines can be used to make thioethers, histidines can beused for linking to a metal ion complex, carboxyl groups can be used forforming amides or esters, amino groups can be used for forming amides,and the like.

The glutenase proteins useful in the practice of the present inventionmay also be isolated and purified in accordance with conventionalmethods from recombinant production systems and from natural sources.Protease production can be achieved using established host-vectorsystems in organisms such as E. coli, S. cerevisiae, P. pastoris,Lactobacilli, Bacilli and Aspergilli. Integrative or self-replicativevectors may be used for this purpose. In some of these hosts, theprotease is expressed as an intracellular protein and subsequentlypurified, whereas in other hosts the enzyme is secreted into theextracellular medium. Purification of the protein can be performed by acombination of ion exchange chromatography, Ni-affinity chromatography(or some alternative chromatographic procedure), hydrophobic interactionchromatography, and/or other purification techniques. Typically, thecompositions used in the practice of the invention will comprise atleast 20% by weight of the desired product, more usually at least about75% by weight, preferably at least about 95% by weight, and fortherapeutic purposes, usually at least about 99.5% by weight, inrelation to contaminants related to the method of preparation of theproduct and its purification. Usually, the percentages will be basedupon total protein.

In one aspect, the present invention provides a purified preparation ofa glutenase. Prior to the present invention, there was no perceived needfor a glutenase that could be ingested by a human or mixed with afoodstuff. Thus, prior to the present invention most glutenases did notexist in a form free of contaminants that could be deleterious to ahuman if ingested. The present invention creates a need for suchglutenase preparations and provides them and methods for preparing them.In a related embodiment, the present invention provides novel foodstuffsthat are derived from gluten-containing foodstuffs but have been treatedto reduce the concentration and amount of the oligopeptides andoligopeptide sequences discovered to be toxic to Celiac Sprue patients.While gluten-free or reduced-gluten content foods have been made, thefoodstuffs of the present invention differ from such foodstuffs not onlyby the manner in which they are prepared, by treatment of the foodstuffwith a glutenase, but also by their content, as the methods of the priorart result in alteration of non-toxic (to Celiac Sprue patients)components of the foodstuff, resulting in a different taste andcomposition. Prior art foodstuffs include, for example, CodexAlimentarius wheat starch, which is available in Europe and has <100 ppmgluten. The starch is usually prepared by processes that take advantageof the fact that gluten is insoluble in water whereas starch is soluble.

In one embodiment of the present invention, a Celiac Sprue patient is,in addition to being provided a glutenase or food treated in accordancewith the present methods, provided an inhibitor of tissuetransglutaminase, an anti-inflammatory agent, an anti-ulcer agent, amast cell-stabilizing agents, and/or and an-allergy agent. Examples ofsuch agents include HMG-CoA reductase inhibitors with anti-inflammatoryproperties such as compactin, lovastatin, simvastatin, pravastatin andatorvastatin; anti-allergic histamine H1 receptor antagonists such asacrivastine, cetirizine, desloratadine, ebastine, fexofenadine,levocetirizine, loratadine and mizolastine; leukotriene receptorantagonists such as montelukast and zafirlukast; COX2 inhibitors such ascelecoxib and rofecoxib; p38 MAP kinase inhibitors such as BIRB-796; andmast cell stabilizing agents such as sodium chromoglycate (chromolyn),pemirolast, proxicromil, repirinast, doxantrazole, amlexanox nedocromiland probicromil.

As used herein, compounds which are “commercially available” may beobtained from commercial sources including but not limited to AcrosOrganics (Pittsburgh Pa.), Aldrich Chemical (Milwaukee Wis., includingSigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park UK), AvocadoResearch (Lancashire U.K.), BDH Inc. (Toronto, Canada), Bionet(Cornwall, U.K.), Chemservice Inc. (West Chester Pa.), Crescent ChemicalCo. (Hauppauge N.Y.), Eastman Organic Chemicals, Eastman Kodak Company(Rochester N.Y.), Fisher Scientific Co. (Pittsburgh Pa.), FisonsChemicals (Leicestershire UK), Frontier Scientific (Logan Utah), ICNBiomedicals, Inc. (Costa Mesa Calif.), Key Organics (Cornwall U.K.),Lancaster Synthesis (Windham N.H.), Maybridge Chemical Co. Ltd.(Cornwall U.K.), Parish Chemical Co. (Orem Utah), Pfaltz & Bauer, Inc.(Waterbury Conn.), Polyorganix (Houston Tex.), Pierce Chemical Co.(Rockford Ill.), Riedel de Haen AG (Hannover, Germany), Spectrum QualityProduct, Inc. (New Brunswick, N.J.), TCI America (Portland Oreg.), TransWorld Chemicals, Inc. (Rockville Md.), Wako Chemicals USA, Inc.(Richmond Va.), Novabiochem and Argonaut Technology.

Compounds useful for co-administration with the glutenases and treatedfoodstuffs of the invention can also be made by methods known to one ofordinary skill in the art. As used herein, “methods known to one ofordinary skill in the art” may be identified though various referencebooks and databases. Suitable reference books and treatises that detailthe synthesis of reactants useful in the preparation of compounds of thepresent invention, or provide references to articles that describe thepreparation, include for example, “Synthetic Organic Chemistry”, JohnWiley & Sons, Inc., New York; S. R. Sandler et al., “Organic FunctionalGroup Preparations,” 2nd Ed., Academic Press, New York, 1983; H. O.House, “Modern Synthetic Reactions”, 2nd Ed., W. A. Benjamin, Inc. MenloPark, Calif. 1972; T. L. Gilchrist, “Heterocyclic Chemistry”, 2nd Ed.,John Wiley & Sons, New York, 1992; J. March, “Advanced OrganicChemistry: Reactions, Mechanisms and Structure”, 4th Ed.,Wiley-Interscience, New York, 1992. Specific and analogous reactants mayalso be identified through the indices of known chemicals prepared bythe Chemical Abstract Service of the American Chemical Society, whichare available in most public and university libraries, as well asthrough on-line databases (the American Chemical Society, Washington,D.C., www.acs.org may be contacted for more details). Chemicals that areknown but not commercially available in catalogs may be prepared bycustom chemical synthesis houses, where many of the standard chemicalsupply houses (e.g., those listed above) provide custom synthesisservices.

The glutenase proteins of the invention and/or the compoundsadministered therewith are incorporated into a variety of formulationsfor therapeutic administration. In one aspect, the agents are formulatedinto pharmaceutical compositions by combination with appropriate,pharmaceutically acceptable carriers or diluents, and are formulatedinto preparations in solid, semi-solid, liquid or gaseous forms, such astablets, capsules, powders, granules, ointments, solutions,suppositories, injections, inhalants, gels, microspheres, and aerosols.As such, administration of the glutenase and/or other compounds can beachieved in various ways, usually by oral administration. The glutenaseand/or other compounds may be systemic after administration or may belocalized by virtue of the formulation, or by the use of an implant thatacts to retain the active dose at the site of implantation.

In pharmaceutical dosage forms, the glutenase and/or other compounds maybe administered in the form of their pharmaceutically acceptable salts,or they may also be used alone or in appropriate association, as well asin combination with other pharmaceutically active compounds. The agentsmay be combined, as previously described, to provide a cocktail ofactivities. The following methods and excipients are exemplary and arenot to be construed as limiting the invention.

For oral preparations, the agents can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

In one embodiment of the invention, the oral formulations compriseenteric coatings, so that the active agent is delivered to theintestinal tract. A number of methods are available in the art for theefficient delivery of enterically coated proteins into the smallintestinal lumen. Most methods rely upon protein release as a result ofthe sudden rise of pH when food is released from the stomach into theduodenum, or upon the action of pancreatic proteases that are secretedinto the duodenum when food enters the small intestine. For intestinaldelivery of a PEP and/or a glutamine specific protease, the enzyme isusually lyophilized in the presence of appropriate buffers (e.g.phosphate, histidine, imidazole) and excipients (e.g. cryoprotectantssuch as sucrose, lactose, trehalose). Lyophilized enzyme cakes areblended with excipients, then filled into capsules, which areenterically coated with a polymeric coating that protects the proteinfrom the acidic environment of the stomach, as well as from the actionof pepsin in the stomach. Alternatively, protein microparticles can alsobe coated with a protective layer. Exemplary films are cellulose acetatephthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulosephthalate and hydroxypropyl methylcellulose acetate succinate,methacrylate copolymers, and cellulose acetate phthalate.

Other enteric formulations comprise engineered polymer microspheres madeof biologically erodable polymers, which display strong adhesiveinteractions with gastrointestinal mucus and cellular linings and cantraverse both the mucosal absorptive epithelium and thefollicle-associated epithelium covering the lymphoid tissue of Peyer'spatches. The polymers maintain contact with intestinal epithelium forextended periods of time and actually penetrate it, through and betweencells. See, for example, Mathiowitz et al. (1997) Nature 386 (6623):410-414. Drug delivery systems can also utilize a core of superporoushydrogels (SPH) and SPH composite (SPHC), as described by Dorkoosh etal. (2001) J Control Release 71(3):307-18.

Gluten detoxification for a gluten sensitive individual can commence assoon as food enters the stomach, since the acidic environment (˜pH 2) ofthe stomach favors gluten solubilization. Introduction of an acid-stablePEP or glutamine-specific protease into the stomach will synergize withthe action of pepsin, leading to accelerated destruction of toxicpeptides upon entry of gluten in the small intestines of celiacpatients. In contrast to a PEP that acts in the small intestine, gastricenzymes need not be formulated with enteric coatings. Indeed, sinceseveral proteases (including the above-mentioned cysteine proteinasefrom barley) self-activate by cleaving the corresponding pro-proteinsunder acidic conditions. In one embodiment of the invention, theformulation comprises a pro-enzyme that is activated in the stomach.

In another embodiment, a microorganism, for example bacterial or yeastculture, capable of producing glutenase is administered to a patient.Such a culture may be formulated as an enteric capsule; for example, seeU.S. Pat. No. 6,008,027, incorporated herein by reference.Alternatively, microorganisms stable to stomach acidity can beadministered in a capsule, or admixed with food preparations.

In another embodiment, the glutenase is admixed with food, or used topre-treat foodstuffs containing glutens. Glutenase present in foods canbe enzymatically active prior to or during ingestion, and may beencapsulated or otherwise treated to control the timing of activity.Alternatively, the glutenase may be encapsulated to achieve a timedrelease after ingestion, e.g. in the intestinal tract.

Formulations are typically provided in a unit dosage form, where theterm “unit dosage form,” refers to physically discrete units suitable asunitary dosages for human subjects, each unit containing a predeterminedquantity of glutenase in an amount calculated sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the unit dosageforms of the present invention depend on the particular complex employedand the effect to be achieved, and the pharmacodynamics associated witheach complex in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are commercially available. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are commercially available. Any compound useful inthe methods and compositions of the invention can be provided as apharmaceutically acceptable base addition salt. “Pharmaceuticallyacceptable base addition salt” refers to those salts which retain thebiological effectiveness and properties of the free acids, which are notbiologically or otherwise undesirable. These salts are prepared fromaddition of an inorganic base or an organic base to the free acid. Saltsderived from inorganic bases include, but are not limited to, thesodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc,copper, manganese, aluminum salts and the like. Preferred inorganicsalts are the ammonium, sodium, potassium, calcium, and magnesium salts.Salts derived from organic bases include, but are not limited to, saltsof primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine,caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine,glucosamine, methylglucamine, theobromine, purines, piperazine,piperidine, N-ethylpiperidine, polyamine resins and the like.Particularly preferred organic bases are isopropylamine, diethylamine,ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

Depending on the patient and condition being treated and on theadministration route, the glutenase may be administered in dosages of0.01 mg to 500 mg/kg body weight per day, e.g. about 20 mg/day for anaverage person. Efficient proteolysis of gluten in vivo for an adult mayrequire at least about 500 units of a therapeutically efficaciousenzyme, usually at least about 1000 units, more usually at least about2000 units, and not more than about 50,000 units, usually not more thanabout 20,000 units, where one unit is defined as the amount of enzymerequired to hydrolyze 1 μmol Cbz-Gly-Pro-pNA (for PEP) orCbz-Gly-Gln-pNA (for a glutamine-specific protease) per min underspecified conditions. Most PEPs have specific activities in the range of5-50 units/mg protein. It will be understood by those of skill in theart that the dose can be raised, but that additional benefits may not beobtained by exceeding the useful dosage. Dosages will be appropriatelyadjusted for pediatric formulation. In children the effective dose maybe lower, for example at least about 0.1 mg, or 0.5 mg. In combinationtherapy involving, for example, a PEP+DPP IV or PEP+DCP I, a comparabledose of the two enzymes may be given; however, the ratio will beinfluenced by the relative stability of the two enzymes toward gastricand duodenal inactivation.

Enzyme treatment of Celiac Sprue is expected to be most efficacious whenadministered before or with meals. However, since food can reside in thestomach for 0.5-2 h, and the primary site of action is expected to be inthe small intestine, the enzyme could also be administered within 1 hourafter a meal.

Optimal gluten detoxification in vivo can also be achieved by combiningan appropriate gastric protease with a PEP that acts upon glutenpeptides in the duodenum, in concert with pancreatic enzymes. This canbe achieved by co-administration of two enzyme doses, e.g. twocapsules/tablets; via co-formulation of the two enzymes in appropriatequantities; etc. Lyophilized duodenal PEP particles or granules can beprotected by a suitable polymeric enteric coating that promotes enzymerelease only in the duodenum. In contrast, release of the gastricprotease will be initiated immediately upon consumption of the dosageform. Combination treatment involving a PEP and a complementarytherapeutic agent, such as an inhibitor of the enzyme tissuetransglutaminase, is also provided.

In some embodiments of the invention, formulations comprise a cocktailof selected proteases. Such combinations may achieve a greatertherapeutic efficacy. In one combination formulation, Flavobacterium PEPand Myxococcus PEP are co-formulated or co-administered, to allow forthe destruction of a broader range of gluten antigenic peptides.Similarly, combination therapy with one or two PEPs from the above listwith an acid-stable PEP or glutamine endoprotease can lead to moreefficient gluten proteolysis in the stomach, thereby simplifying thetask of gluten assimilation in the upper small intestine.

In another embodiment, the formulation or administration protocolcombines a protease product and an inhibitor of transglutaminase 2(TG2). Such formulations may have additional protection from glutenmediated enteropathy, as TG2 has been shown to have a significantpro-inflammatory effect on gluten peptides in the celiac gut. Inparticular, TG2 inhibitors containing halo-dihydroisoxazole,diazomethylketone or dioxoindole moieties are useful for this purpose.

In another embodiment, the protease or protease cocktail is administeredand/or formulated with an anti-inflammatory agent, e.g. a statin; p38MAP kinase inhibitor; anti-TNFα agent; etc.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific enzyme, the severity of the symptoms and thesusceptibility of the subject to side effects. Some of the glutenasesare more potent than others. Preferred dosages for a given enzyme arereadily determinable by those of skill in the art by a variety of means.A preferred means is to measure the physiological potency of a givencompound.

Other formulations of interest include formulations of DNA encodingglutenases of interest, so as to target intestinal cells for geneticmodification. For example, see U.S. Pat. No. 6,258,789, hereinincorporated by reference, which discloses the genetic alteration ofintestinal epithelial cells.

The methods of the invention are used to treat foods to be consumed orthat are consumed by individuals suffering from Celiac Sprue and/ordermatitis herpetiformis by delivering an effective dose of glutenase.If the glutenase is administered directly to a human, then the activeagent(s) are contained in a pharmaceutical formulation. Alternatively,the desired effects can be obtained by incorporating glutenase into foodproducts or by administering live organisms that express glutenase, andthe like. Diagnosis of suitable patients may utilize a variety ofcriteria known to those of skill in the art. A quantitative increase inantibodies specific for gliadin, and/or tissue transglutaminase isindicative of the disease. Family histories and the presence of the HLAalleles HLA-DQ2 [DQ(a1*0501, b1*02)] and/or DQ8 [DQ(a1*0301, b1*0302)]are indicative of a susceptibility to the disease.

The therapeutic effect can be measured in terms of clinical outcome orcan be determined by immunological or biochemical tests. Suppression ofthe deleterious T-cell activity can be measured by enumeration ofreactive Th1 cells, by quantitating the release of cytokines at thesites of lesions, or using other assays for the presence of autoimmune Tcells known in the art. Alternatively, one can look for a reduction insymptoms of a disease.

Various methods for administration may be employed, preferably usingoral administration, for example with meals. The dosage of thetherapeutic formulation will vary widely, depending upon the nature ofthe disease, the frequency of administration, the manner ofadministration, the clearance of the agent from the host, and the like.The initial dose can be larger, followed by smaller maintenance doses.The dose can be administered as infrequently as weekly or biweekly, ormore often fractionated into smaller doses and administered daily, withmeals, semi-weekly, or otherwise as needed to maintain an effectivedosage level.

This application is related to U.S. Provisional 60/565,668, filed Apriol26, 2004; to U.S. Provisional application 60/357,238 filed Feb. 14,2002; to U.S. Provisional Application 60/380,761 filed May 14, 2002; toU.S. Provisional Application 60/392,782 filed Jun. 28, 2002; and to U.S.Provisional application No. 60/422,933, filed Oct. 31, 2002, to U.S.Provisional Application 60/428,033, filed Nov. 20, 2002, to U.S.Provisional Application 60/435,881, filed Dec. 20, 2002, and to U.S.Ser. No. 10/367,405, filed Feb. 14, 2004, each of which are hereinspecifically incorporated by reference.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of the invention or to represent that the experiments below areall or the only experiments performed. Efforts have been made to ensureaccuracy with respect to numbers used (e.g., amounts, temperature, andthe like), but some experimental errors and deviations may be present.Unless indicated otherwise, parts are parts by weight, molecular weightis weight average molecular weight, temperature is in degreesCentigrade, and pressure is at or near atmospheric.

Example 1 Detection of Immunodominant Peptides from Gliadin and Enzymesthat Degrade them

The following examples describe the discovery and characterization of asmall number of immunodominant peptides from gliadin, which account formost of the stimulatory activity of dietary gluten on intestinal andperipheral T lymphocytes found in Celiac Sprue patients. The proteolytickinetics of these immunodominant peptides were analyzed at the smallintestinal surface. Brush border membrane vesicles from adult ratintestines were used to show that these proline-glutamine-rich peptidesare exceptionally resistant to enzymatic processing, and that dipeptidylpeptidase IV and dipeptidyl carboxypeptidase are the rate-limitingenzymes in their digestion. Supplementation of the brush border membranewith trace quantities of a bacterial prolyl endopeptidase leads to rapiddestruction of these gliadin peptides. These results provide the basisfor enzyme-mediated therapies for treating food for provision to CeliacSprue patients, and for treating such patients directly that offerdistinct advantages over the only current therapeutic option, which isstrict exclusion of gluten containing food.

To investigate the digestion of gluten, liquid chromatography coupledmass spectroscopy analysis (LC-MS-MS) was utilized to investigate thepathways and associated kinetics of hydrolysis of immunodominant gliadinpeptides treated with rat BBM preparations. Because the rodent is anexcellent small animal model for human intestinal structure andfunction, rat BBM was chosen as a suitable model system for thesestudies.

BBM fractions were prepared from rat small intestinal mucosa asdescribed in Ahnen et al. (1982) J. Biol. Chem. 257, 12129-35. Thespecific activities of the known BB peptidases were determined to be 127μU/μg for Aminopeptidase N (APN, EC 3.4.11.2), 60 μU/μg for dipeptidylpeptidase IV (DPP IV, EC 3.4.14.5), and 41 μU/μg for dipeptidylcarboxypeptidase (DCP, EC 3.4.15.1) using standard assays. No prolineaminopeptidase (EC 3.4.11.5) or prolyl endopeptidase activity (PEP, EC3.4.21.26) activity was detectable (<5 μU/μg). Alkaline phosphatase andsucrase were used as control BBM enzymes with activities of 66 μU/μg and350 μU/μg, respectively.

BBM fractions were partially purified from the small intestinal mucosaof adult female rats maintained on an ad libitum diet of wheat-basedstandard rodent chow. Total protein content was determined by a modifiedmethod of Lowry with BSA as a standard. Alkaline phosphatase activitywas determined with nitrophenyl phosphate. Sucrase activity was measuredusing a coupled glucose assay. DPP IV, proline aminopeptidase and APNwere assayed continuously at 30° C. in 0.1 M Tris-HCl, pH 8.0,containing 1 mM of the p-nitroanilides (ε=8,800 M⁻¹ cm⁻¹) Gly-Pro-pNA,Pro-pNA or Leu-pNA, the latter in additional 1% DMSO to improvesolubility. DCP activity was measured in a 100 μl reaction as therelease of hippuric acid from Hip-His-Leu. PEP activity was determinedcontinuously with 0.4 mM Z-Gly-Pro-pNA in PBS:H₂O:dioxane (8:1.2:0.8) at30° C. One unit is the consumption of 1 μmol substrate per minute.

DPP IV and DCP are both up-regulated by a high proline content in thediet. However, APN activity using standard substrates was found to behigher than DPP IV even when fed extreme proline rich diets. Also,although a higher DCP vs. CPP activity has been observed with the modelpeptide Z-GPLAP at saturating concentrations, a difference in Km valuescould easily account the reversed ratio measured. The amount of 100 μMwas chosen as the initial peptide concentration, because non-saturatingkinetics (k_(cat)/K_(m)) were considered to be physiologically morerelevant than the maximal rates of hydrolysis (k_(cat)).

Proteolysis with the BBM preparation was investigated using the peptide(SEQ ID NO:1) QLQPFPQPQLPY, a product of chymotryptic digestion of α-9gliadin (Arentz-Hansen et al. (2000) J. Exp. Med. 191, 603-12). Thispeptide has been shown to stimulate proliferation of T cells isolatedfrom most Celiac Sprue patients, and hence is considered to possess animmunodominant epitope. It was subjected to BBM digestion, followed byLC-MS-MS analysis. A standard 50 μl digestion mixture contained 100 μMof synthetic peptide, 10 μM tryptophan and Cbz-tryptophan as internalstandards, and resuspended BBM preparations with a final protein contentof 27 ng/μl and exogenous proteins, as indicated, in phosphate bufferedsaline. After incubation at 37° C. for the indicated time, the enzymeswere inactivated by heating to 95° C. for 3 minutes. The reactionmixtures were analyzed by LC-MS (SpectraSystem, ThermoFinnigan) using aC18 reversed phase column (Vydac 218TP5215, 2.1×150 mm) withwater:acetonitrile:formic acid (0.1%):trifluoroacetic acid (0.025%) asthe mobile phase (flow: 0.2 ml/min) and a gradient of 10% acetonitrilefor 3 minutes, 10-20% for 3 minutes, 20-25% for 21 minutes followed by a95% wash. Peptide fragments in the mass range of m/z=300-2000 weredetected by electrospray ionization mass spectroscopy using a LCQ iontrap and their identities were confirmed by MSMS fragmentation patterns.

While the parent peptide (SEQ ID NO:1) QLQPFPQPQLPY disappeared with anapparent half life of 35 min, several intermediates were observed toaccumulate over prolonged periods (FIG. 1A). The MS intensities(m/z=300-2000 g/mol) and UV₂₈₀ absorbances of the parent peptides (SEQID NO:1) QLQPFPQPQLPY and (SEQ ID NO:3) PQPQLPYPQPQLPY were found todepend linearly on concentration in the range of 6-100 μM. The referencepeptides (SEQ ID NO:4) PQPQLPYPQPQLP, (SEQ ID NO:5) QLQPFPQPQLP, (SEQ IDNO:6) QPQFPQPQLPY and (SEQ ID NO:7) QPFPQPQLP were generatedindividually by limited proteolysis of the parent peptides with 10 μg/mlcarboxypeptidase A (C-0261, Sigma) and/or 5.9 μg/ml leucineaminopeptidase (L-5006, Sigma) for 160 min at 37° C. and analyzed byLC-MS as in FIG. 1.

Indeed, the subsequent processing of the peptide was substantiallyretarded (FIG. 1B). The identities of the major intermediates wereconfirmed by tandem MS, and suggested an unusually high degree ofstability of the (SEQ ID NO:8) PQPQLP sequence, a common motif in T cellstimulating peptides. Based on this data and the known amino acidpreferences of the BBM peptidases, the digestive breakdown of (SEQ IDNO:1) QLQPFPQPQLPY was reconstructed, as shown in the insert of FIG. 1B.The preferred pathway involves serial cleavage of the N-terminalglutamine and leucine residues by aminopeptidase N (APN), followed byremoval of the C-terminal tyrosine by carboxypeptidase P (CPP) andhydrolysis of the remaining N-terminal QP-dipeptide by DPP IV. As seenin FIG. 1B, the intermediate (SEQ ID NO:6) QPFPQPQLPY (formed by APNattack on the first two N-terminal residues) and its derivatives areincreasingly resistant to further hydrolysis. Because the high prolinecontent seemed to be a major cause for this proteolytic resistance,digestion was compared with a commercially available non-proline controlpeptide (SEQ ID NO:9) RRLIEDNEYTARG (Sigma, St. Louis, Mo.). Initialhydrolysis was much faster (t_(1/2)=10 min). More importantly, digestiveintermediates were only transiently observed and cleared completelywithin one hour, reflecting a continuing high specificity of the BBM forthe intermediate peptides.

Because the three major intermediate products (SEQ ID NO:10) QPFPQPQLPY,(SEQ ID NO:7) QPFPQPQLP, (SEQ ID NO:11) FPQPQLP) observed during BBMmediated digestion of (SEQ ID NO:1) QLQPFPQPQLPY are substrates for DPPIV, the experiment was repeated in the presence of a 6-fold excessactivity of exogenous fungal DPP IV. Whereas the relatively rapiddecrease of the parent peptide and the intermediate levels of (SEQ IDNO:5) QLQPFPQPQLP were largely unchanged, the accumulation of DPP IVsubstrates was entirely suppressed, and complete digestion was observedwithin four hours. (FIG. 1B, open bars).

To investigate the rate-limiting steps in BBM mediated digestion ofgliadin peptides from the C-terminal end, another known immunodominantpeptide derived from wheat α-gliadin, (SEQ ID NO:3) PQPQLPYPQPQLPY, wasused. Although peptides with N-terminal proline residues are unlikely toform in the small intestine (none were observed during BBM digestion of(SEQ ID NO:1) QLQPFPQPQLPY, FIG. 1A), they serve as a useful model forthe analysis of C-terminal processing, because the N-terminal end ofthis peptide can be considered proteolytically inaccessible due tominimal proline aminopeptidase activity in the BBM. As shown in FIG. 2,this peptide is even more stable than (SEQ ID NO:1) QLQPFPQPQLPY. Inparticular, removal of the C-terminal tyrosine residue bycarboxypeptidase P (CPP) is the first event in its breakdown, and morethan four times slower than APN activity on (SEQ ID NO:1) QLQPFPQPQLPY(FIG. 1B). The DCP substrate (SEQ ID NO:4) PQPQLPYPQPQLP emerges as amajor intermediate following carboxypeptidase P catalysis, and is highlyresistant to further digestion, presumably due to the low level ofendogenous DCP activity naturally associated with the BBM. To confirmthe role of DCP as a rate-limiting enzyme in the C-terminal processingof immunodominant gliadin peptides, the reaction mixtures weresupplemented with rabbit lung DCP. Exogenous DCP significantly reducedthe accumulation of (SEQ ID NO:4) PQPQLPYPQPQLP after overnightincubation in a dose dependent manner. Conversely, the amount ofaccumulated (SEQ ID NO:4) PQPQLPYPQPQLP increased more than 2-fold inthe presence of 10 μM of captopril, a DCP-specific inhibitor, ascompared with unsupplemented BBM.

Together, the above results demonstrate that (i) immunodominant gliadinpeptides are exceptionally stable toward breakdown catalyzed by BBMpeptidases, and (ii) DPP IV and especially DCP are rate-limiting stepsin this breakdown process at the N- and C-terminal ends of the peptides,respectively. Because BBM exopeptidases are restricted to N- orC-terminal processing, it was investigated if generation of additionalfree peptide ends by pancreatic enzymes would accelerate digestion. Ofthe pancreatic proteases tested, only elastase at a high(non-physiological) concentration of 100 ng/μl was capable ofhydrolyzing (SEQ ID NO:3) PQPQLPYPQPQ^(↓)LPY. No proteolysis wasdetected with trypsin or chymotrypsin.

Alerted by the high proline content as a hallmark of most immunogenicgliadin peptides, a proline-specific endopeptidase was tested for thegeneration of new, free peptide termini. A literature search onavailable proteases led to the identification of prolyl endopeptidase(PEP) from Flavobacterium meningosepticum, which is specific for theC-terminal cleavage of prolines and readily available from recombinantsources (Yoshimoto et al. (1991) J. Biochem. 110, 873-8). The stable(SEQ ID NO:4) PQPQLPYPQPQLP intermediate was digested with BBM in thepresence of exogenous PEP. FIG. 3 shows the dose dependent accelerationof (SEQ ID NO:4) PQPQLPYPQPQLP digestion with increasing PEPconcentration. As little as 3.5 pg PEP/27 ng BBM protein was sufficientto double the extent of proteolysis of this gliadin fragment compared toincubation with BBM alone. In comparison, other commonly used proteaseslike papain, bromelain or porcine elastase were much less efficient,requiring 30-fold (papain) or 3000-fold (bromelain, elastase) higheramounts of enzyme compared to PEP to give similar results. Theirproteolysis was restricted to the cleavage of the Gln⁴-Leu⁵ and/orGln¹¹-Leu¹² bonds.

Prolyl endopeptidase (EC 3.4.21.26) had a preference for the Pro⁸-Gln⁹and to a lesser extent the Pro⁶-Tyr⁷ bond of the (SEQ ID NO:4)PQPQLP^(↓)YP^(↓)QPQLP peptide. A similar preferential cleavage was foundfor (SEQ ID NO:1) QLQPFP^(↓)QPQLPY. This is in agreement with thepreference of this prolyl endopeptidase for a second proline in the S2′position (Bordusa and Jakubke (1998) Bioorg. Med. Chem. 6, 1775-80).Based on this P^(↓)XP motif and on the present data, up to 16 new, majorcleavage sites can be predicted in the α2-gliadin sequence, a majorsource of immunodominant epitopes identified thus far upon PEPtreatment. All of them are located in the critical N-terminal part. Theinternal cleavage by PEP can be expected to generate additional(otherwise inaccessible) substrates for DPP IV and DCP therebycomplementing the natural assimilation process of gliadins by the BBM.Thus, the specificity of prolyl endopeptidase is ideally suited fordetoxification of persistent immunoactive gliadin peptides in CeliacSprue.

The above data demonstrates that proline-rich gliadin peptides areextraordinarily resistant to digestion by small intestinal endo- andexopeptidases, and therefore are likely to accumulate at highconcentrations in the intestinal cavity after a gluten rich meal. Thepathological implication of digestive resistance is strengthened by theobserved close correlation of proline content and celiac toxicity asobserved in the various common cereals (Schuppan (2000) Gastroenterology119, 234-42). This analysis of the digestive pathways of immunodominantpeptides also provides a mechanism for determining whether enzymescapable of accelerating this exceptionally slow process can betherapeutically useful in the Celiac Sprue diet.

Addition of exogenous DPP IV and DCP can compensate for theintrinsically slow proline processing by the BBM, although both enzymesrely on efficient generation of free N- and C-termini by endoproteolyticcleavage. In a preferred embodiment, a soluble bacterial prolylendopeptidase (PEP) is used, which was shown to be extremely efficientat hydrolyzing the proline-rich gliadin fragments. Although PEP isexpressed in human brain, lung, kidney and intestine, no such activityhas been reported in the brush border.

Supplementation of the Celiac Sprue diet with bioavailable PEP (with orwithout DPP IV and/or DCP), by virtue of facilitating gliadin peptidecleavage to non-toxic and/or digestible fragments, is useful inattenuating or eliminating the inflammatory response to gluten. Such atreatment regimen is analogous to the enzyme therapy treatment used totreat lactose intolerance, where orally administered lactase iseffective in cleaving and thereby detoxifying the lactose in milkproducts. Prolyl endopeptidases are widely distributed inmicroorganisms, plants and animals and have been cloned from Aeromonashydrophyla (Kanatani et al. (1993) J. Biochem. 113, 790-6); Pyrococcusfurious (Robinson et al. (1995) Gene 152, 103-6) and from pig brain(Rennex et al. (1991) Biochemistry 30, 2195-2030). These isozymesconstitute alternative detoxifying peptidases. Furthermore, the prolylendopeptidase used in this study is readily amenable to proteinengineering by directed evolution. Thus, optimization of PEP specificitytowards immunogenic gliadin peptides can be achieved.

Example 2 Further Characterization of Immunodominant Gliadin Peptidesand Means for their Digestion

It has long been known that the principal toxic components of wheatgluten are a family of closely related Pro-Gln rich proteins calledgliadins. Peptides from a short segment of α-gliadin appear to accountfor most of the gluten-specific recognition by CD4+ T cells from CeliacSprue patients. These peptides are substrates of tissue transglutaminase(tTGase), the primary auto-antigen in Celiac Sprue, and the products ofthis enzymatic reaction bind to the class II HLA DQ2 molecule. Thisexample describes a combination of in vitro and in vivo animal and humanstudies used to characterize this “immunodominant” region of α-gliadinas part of an unusually long proteolytic product generated by thedigestive process that: (a) is exceptionally resistant to furtherbreakdown by gastric, pancreatic and intestinal brush border proteases;(b) is the highest specificity substrate of human tissuetransglutaminase (tTGase) discovered to date; (c) contains at least sixoverlapping copies of epitopes known to be recognized by patient derivedT cells; (d) stimulates representative T cell clones that recognizethese epitopes with sub-micromolar efficacy; and (e) has homologs inproteins from all toxic foodgrains but no homologs in non-toxicfoodgrain proteins. In aggregate, these findings demonstrate that theonset of symptoms upon gluten exposure in the Celiac Sprue patient canbe traced back to a small segment of α-gliadin. Finally, it is shownthat this “super-antigenic” long peptide can be detoxified in vitro andin vivo by treatment with bacterial prolyl endopeptidase, providing apeptidase therapy for Celiac Sprue.

Identification of stable peptides from gastric protease, pancreaticprotease and brush border membrane peptidase catalyzed digestion ofrecombinant α2-gliadin: The protein α2-gliadin, a representativeα-gliadin (Arentz-Hansen et al. (2000) Gut 46:46), was expressed inrecombinant form and purified from E. coli. The α2-gliadin gene wascloned in pET28a plasmid (Novagen) and transformed into the expressionhost BL21(DE3) (Novagen). The transformed cells were grown in 1-litercultures of LB media containing 50 μg/ml of kanamycin at 37° C. untilthe OD600 0.6-1 was achieved. The expression of α2-gliadin protein wasinduced with the addition of 0.4 mM isopropyl β-D-thiogalactoside(Sigma) and the cultures were further incubated at 37° C. for 20 hours.The cells expressing the recombinant α2-gliadin were centrifuged at 3600rpm for 30 minutes. The pellet was resuspended in 15 ml of disruptionbuffer (200 mM sodium phosphate; 200 mM NaCl; 2.5 mM DTT; 1.5 mMbenzamidine; 2.5 mM EDTA; 2 mg/L pepstatin; 2 mg/L leupeptin; 30% v/vglycerol) and lysed by sonication (1 minute; output control set to 6).After centrifugation at 45000 g for 45 min, the supernatant wasdiscarded and the pellet containing gliadin protein was resuspended in50 ml of 7 M urea in 50 mM Tris (pH=8.0). The suspension was againcentrifuged at 45000 g for 45 min and the supernatant was harvested forpurification. The supernatant containing α2-gliadin was incubated with 1ml of nickel-nitrilotriacetic acid resin (Ni-NTA; Qiagen) overnight andthen batch-loaded on a column with 2 ml of Ni-NTA. The column was washedwith 7M urea in 50 mM Tris (pH=8.0), and α2-gliadin was eluted with 200mM imidazole, 7 M urea in 50 mM Tris (pH=4.5). The fractions containingα2-gliadin were pooled into a final concentration of 70% ethanolsolution and two volumes of 1.5M NaCl were added to precipitate theprotein. The solution was incubated at 4 PC overnight and the finalprecipitate was collected by centrifugation at 45000 g for 30 min,rinsed in water, and re-centrifuged to remove the urea. The finalpurification step of the α-2 gliadin was developed with reverse-phaseHPLC. The Ni-NTA purified protein fractions were pooled in 7 M ureabuffer and injected to a Vydac (Hesperia, Calif.) polystyrenereverse-phase column (i.d. 4.6 mm×25 cm) with the starting solvent (30%of solvent B: 1:1 HPLC-grade acetonitrile/isopropanol:0.1% TFA). SolventA was an aqueous solution with 0.1% TFA. The separation gradientextended from 30-100% of solvent B over 120 min at a flow rate of 0.8ml/min.

TABLE 2 Amount of Peptides Digested after 15 hours 33-mer Control AControl B H1P0 <20% >90% >90% H2P0 <20% >61% >85% H3P0 <20% >87% >95%H4P0 <20% >96% >95% H5P0 <20% >96% >95%

The purity of the recombinant gliadin was >95%, which allowed for facileidentification and assignment of proteolytic products by LC-MS/MS/UV.Although many previous studies utilized pepsin/trypsin treated gliadins,it was found that, among gastric and pancreatic proteases, chymotrypsinplayed a major role in the breakdown of α2-gliadin, resulting in manysmall peptides from the C-terminal half of the protein and a few longer(>8 residues) peptides from the N-terminal half, the most noteworthybeing a relatively large fragment, the 33-mer (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (residues 57-89). This peptide was ofparticular interest for two reasons: (a) whereas most other relativelystable proteolytic fragments were cleaved to smaller fragments when thereaction times were extended, the 33-mer peptide remained intact despiteprolonged exposure to proteases; and (b) three distinct patient-specificT cell epitopes identified previously are present in this peptide,namely, (SEQ ID NO:20) PFPQPQLPY, (SEQ ID NO:21) PQPQLPYPQ (3 copies),and (SEQ ID NO:22) PYPQPQLPY (2 copies).

To establish the physiological relevance of this peptide, compositegastric/pancreatic enzymatic digestion of α2 gliadin was then examined.As expected, enzymatic digestion with pepsin (1:100 w/w ratio), trypsin(1:100), chymotrypsin (1:100), elastase (1:500) and carboxypeptidase(1:100) was quite efficient, leaving behind only a few peptides longerthan 9 residues (the minimum size for a peptide to show class II MHCmediated antigenicity) (FIG. 4). In addition to the above-mentioned33-mer, the peptide (SEQ ID NO:23) WQIPEQSR was also identified, and wasused as a control in many of the following studies. The stability of the33-mer peptide can also be appreciated when comparing the results of asimilar experiment using myoglobin (another common dietary protein).Under similar proteolytic conditions, myoglobin is rapidly broken downinto much smaller products. No long intermediate is observed toaccumulate.

The small intestinal brush-border membrane (BBM) enzymes are known to bevital for breaking down any remaining peptides from gastric/pancreaticdigestion into amino acids, dipeptides or tripeptides for nutritionaluptake. Therefore a comprehensive analysis of gliadin metabolism alsorequired investigations into BBM processing of gliadin peptides ofreasonable length derived from gastric and pancreatic proteasetreatment. BBM fractions were prepared from rat small intestinal mucosa.The specific activities of known BBM peptidases were verified to bewithin the previously reported range. Whereas the half-life ofdisappearance of WQIPEQSR was ˜60 min. in the presence of 12 ng/μl BBMprotein, the half-life of (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF digestion was >20 h. Therefore, thelatter peptide remains intact throughout the digestive process in thestomach and upper small intestine, and is poised to act as a potentialantigen for T cell proliferation and intestinal toxicity in geneticallysusceptible individuals.

Verification of proteolytic resistance of the 33-mer gliadin peptidewith brush border membrane preparations from human intestinal biopsies:to validate the conclusions reached as described in Example 1, whichdescribes studies with rat BBM preparations, in the context of humanintestinal digestion, BBM preparations were prepared from a panel ofadult human volunteers, one of whom was a Celiac Sprue patient inremission, while the rest were found to have normal intestinalhistology. (SEQ ID NO:12) LQLQPFPQPQLPYPQPQL PYPQPQLPYPQPQPF, (SEQ IDNO:1) QLQPFPQPQLPY (an internal sequence from the 33-mer used as acontrol), WQIPEQSR and other control peptides (100 μM) were incubatedwith BBM prepared from each human biopsy (final aminopeptidase Nactivity of 13 μU/μl) at 37° C. for varying time periods. While (SEQ IDNO:1) QLQPFPQPQLPY, (SEQ ID NO:23) WQIPEQSR and other control peptideswere completely proteolyzed within 1-5 h, the long peptide remainedlargely intact for at 19 hours. These results confirm the equivalencebetween the rat and human BBM for the purpose of this study. Moreover,these results indicate that the methods, foodstuffs, and other reagentsof the invention can be used in humans not known to have Celiac Sprue toimprove digestion and reduce any ill effects of the long peptide.

Verification of proteolytic resistance of the 33-mer gliadin peptide inintact animals: The proteolytic resistance of the 33-mer gliadinpeptide, observed in vitro using BBM from rats and humans, was confirmedin vivo using a perfusion protocol in intact adult rats (Smithson andGray (1977) J. Clin. Invest. 60:665). Purified peptide solutions wereperfused through a 15-20 cm segment of jejunum in a sedated rat with aresidence time of 20 min, and the products were collected and subjectedto LC-MS analysis. Whereas >90% of (SEQ ID NO:1) QLQPFPQPQLPY wasproteolyzed in the perfusion experiment, most of the 33-mer gliadinpeptide remained intact. These results demonstrate that the 33-merpeptide is very stable as it is transported through the mammalian uppersmall intestine. The data is shown in FIG. 5.

The 33-mer gliadin peptide is an excellent substrate for tTGase, and theresulting product is a highly potent activator of patient-derived Tcells: studies have demonstrated that regiospecific deamidation ofimmunogenic gliadin peptides by tTGase increases their affinity forHLA-DQ2 as well as the potency with which they activate patient-derivedgluten-specific T cells. It has been shown that the specificity oftTGase for certain short antigenic peptides derived from gliadin ishigher than its specificity toward its physiological target site infibronectin; for example, the specificity of tTGase for the α-gliadinderived peptide (SEQ ID NO:3) PQPQLPYPQPQLPY is 5-fold higher than thatfor its target peptide sequence in fibrinogen, its natural substrate.The kinetics and regiospecificity of deamidation of the 33-mer α-gliadinpeptide identified as above were therefore measured. The k_(cat)/K_(M)was higher than that reported for any peptide studied thus far:kcat/KM=440 min-1 mM-1 for (SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF compared to kcat/KM=82 min-1 mM-1 for PQPQLPY andkcat/KM=350 min-1 mM-1 for (SEQ ID NO:3) PQPQLPYPQPQLPY.

Moreover, LC-MS-MS analysis revealed that the peptide (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF was selectively deamidated by tTGaseat the underlined residues. Because tTGase activity is associated withthe brush border membrane of intestinal enterocytes, it is likely thatdietary uptake of even small quantities of wheat gluten will lead to thebuild-up of sufficient quantities of this 33-mer gliadin peptide in theintestinal lumen so as to be recognized and processed by tTGase.

Structural characteristics of the 33-mer gliadin peptide and itsnaturally occurring homologs: Sequence alignment searches using BLASTPin all non-redundant protein databases revealed several homologs(E-value <0.001) of the 33-mer gliadin peptide, shown in FIG. 6.Interestingly, foodgrain derived homologs were only found in gliadins(from wheat), hordeins (from barley) and secalins (from rye), all ofwhich have been proven to be toxic to Celiac Sprue patients. Nontoxicfoodgrain proteins, such as avenins (in oats), rice and maize, do notcontain homologous sequences to the 33-mer gliadin. In contrast, aBLASTP search with the entire 2-gliadin sequence identified foodgrainprotein homologs from both toxic and nontoxic proteins. Based onavailable information regarding the substrate specificities of gastric,pancreatic and BBM proteases and peptidases, it is believed that,although most gluten homologs to the 33-mer gliadin peptide containedmultiple proteolytic sites and are therefore unlikely to be completelystable toward digestion, several sequences from wheat, rye and barleyare expected to be resistant to gastric and intestinal proteolysis. Thestable peptide homologs to the 33-mer a2-gliadin peptide are (SEQ IDNO:24) QPQPFPPQLPYPQTQPFPPQQPYPQPQPQYPQPQ (from α1- and α6-gliadins);(SEQ ID NO:25) QQQPFPQQPIPQQPQPYPQQPQPYPQQPFPPQQPF (from B1 hordein);(SEQ ID NO:26) QPFPQPQQTFPQQPQLPFPQQPQQPFPQPQ (from γ-gliadin); (SEQ IDNO:27) QPFPQPQQPTPIQPQQPFPQRPQQPFPQPQ (from ω-secalin). These stablepeptides are all located at the N-terminal region of the correspondingproteins. The presence of proline residues after otherwise cleavableresidues in these peptides would contribute to their proteolyticstability.

Bacterial prolyl endopeptidase rapidly detoxifies the 33-mer gliadinpeptide: The abundance and location of proline residues is a crucialfactor contributing to the resistance the 33-mer gliadin peptide towardgastrointestinal breakdown. In accordance with the methods of theinvention, a prolyl endopeptidase can catalyze breakdown of thispeptide, thereby diminishing its toxic effects. Preliminary in vitrostudies with short gliadin peptides and the prolyl endopeptidase (PEP)from F. meningosepticum demonstrate this aspect of the invention. Theability of this PEP to clear the 33-mer gliadin peptide was evaluatedvia in vitro and in vivo experiments. Using both rat BBM andco-perfusion of the peptide and PEP in intact rat intestines, thisdetoxification was demonstrated. The results are shown in FIG. 7.Together these results highlight the potential of detoxifying gluten inCeliac Sprue patients by peptidase therapy.

Although gluten proteins from foodgrains such as wheat, rye and barleyare central components of a nutritious diet, they can be extremely toxicfor patients suffering from Celiac Sprue. To elucidate the structuralbasis of gluten toxicity in Celiac Sprue, comprehensive proteolyticanalysis was performed on a representative recombinant gliadin underphysiologically relevant conditions. An unusually long andproteolytically stable peptide product was discovered, whosephysiological relevance was confirmed by studies involving brush bordermembrane proteins from rat and human intestines as well as intestinalperfusion assays in live rats. In aggregate, these data demonstrate thatthis peptide and its homologs found in other wheat, rye and barleyproteins contribute significantly to the inflammatory response todietary wheat in Celiac Sprue patients.

The absence of satisfactory animal models for Celiac Sprue implies thatthe pivotal pathogenic nature of the gluten peptides identified in thisstudy can only be verified in human patients. The results abovedemonstrate that the deleterious effects of gluten ingestion by CeliacSprue patients can be amelioriated by enzyme treatment of glutencontaining foods. Specifically, co-administration of a bioavailable formof a suitable prolyl endopeptidase with dietary gluten attenuate itstoxicity by cleaving the stable 33-mer peptide into non-immunogenicproducts. Given the absence of a satisfactory therapeutic option forCeliac Sprue and the notorious difficulty associated with long-termmaintenance of a gluten-free diet, the peptidase therapies of thepresent invention provides an alternative to strict abstinence for therapidly growing numbers of individuals affected by this disease.

Example 3 Comparison of PEP Activities

To gain insight into the similarities and differences between naturallyoccurring prolyl endopeptidases, we have systematically compared theproperties of three homologous PEPs from different bacterial sources.Our studies have utilized two known recombinant PEPs from Flavobacteriummeningosepticum (FM) and Sphingomonas capsulata (SC), respectively, anda novel PEP from Myxococcus xanthus (MX) that we have expressed for thefirst time as a heterologous recombinant protein. The enzymaticactivities of these PEPs were quantitatively analyzed versus modelsubstrates as well as two gluten-derived peptides with potentialrelevance to Celiac Sprue pathogenesis. In particular, we have probedthe influence of substrate chain length, pH, pancreatic proteases andintestinal brush border peptidases on the activity of each PEP. Both invivo and ex vivo experiments were performed as part of these studies.

Experimental Procedures

Cloning of PEP Genes. The PEP genes were amplified from the genomic DNAfrom the corresponding bacterial strains (F. meningosepticum: ATCC13253; S. capsulata: ATCC 14666; M. xanthus: ATCC 25232). The sequenceof the putative MX PEP is available from the NCBI database (Locus IDAAD31004). Oligonucleotides used for PCR amplification included: (SEQ IDNO:37) (1) FM first half: 5′-AAC CAA TCA TAT GAA GTA CAA CAA ACT TTC TGTG (NdeI), (SEQ ID NO:38) 5′-GAT AAA AAC GGA AAG CTT GTA AGG GC(HindIII); FM second half: (SEQ ID NO:39) 5′-GCC CTT ACA AGC TTT CCG TTTTTA TC (HindIII) and (SEQ ID NO:40) 5′-CCC TTA ATT TTC AAA TTT TAG CTCGAG TTT ATG ATT TAT A (SacI); (2) SC first half: (SEQ ID NO:41) 5′-AGGATA TCC ATA TGA AGA ACC GCT TGT GG (NdeI), (SEQ ID NO:42) 5′-GAC AAC CTCGAA TCC GTC GGC ATT G (HinfI); SC second half: (SEQ ID NO:43) 5′-CAA TGCCGA CGG ATT CGA GGT TGT C (HinfI), (SEQ ID NO:44) 5′-CGC GGG GAC CTC GAGTAG AAA CTG (SacI); (3) MX: (SEQ ID NO:45) 5′-CT CCC CAT ATG TCC TAC CCGGCG ACC (NdeI) and (SEQ ID NO:46) 5′-GTG GCG GCG CAG GGC CGC AAG CTT CCCAAG CG (HindIII). The amplified genes were cloned into a pET28b plasmid(Novagen).

Expression and Purification of PEPs. Expression plasmids were introducedvia transformation into BL21(DE3) cells. Transformants grown at 37° C.,and induced in the presence of 100 μM IPTG at 22° C. overnight. Lowtemperature induction was found to improve the yield of active enzyme.All purification steps were performed at 4° C. unless noted otherwise.Since FM and SC PEP enzymes naturally possess a signal sequence, theyare secreted into the periplasmic space of E. coli. A modified osmoticshock protocol (EMD Biosciences, CA) was therefore used to obtain anenriched protein lysate containing either PEP. Cell pellets (4 L ofculture) were resuspended in 30 ml of 30 mM Tris-HCl, pH 8, 20% sucroseand 1 mM EDTA, and stirred slowly at room temperature for 10 min. Thesuspension was centrifuged at 10,000 g for 15 min, and the cell pelletwas resuspended in ice-cold dH₂O and stirred slowly on ice for 10 min.The shocked cells were then centrifuged again at 40,000-50000 g for 30min. The supernatant containing the periplasmic proteins was treated for1-2 h with 1 M NaCl solution (to a final concentration of 300 mM NaCl),1 M imidazole solution (to a final concentration 5 mM imidazole) and 1ml of Ni-NTA resin (Qiagen, CA). The crude protein was then loaded ontoa column containing additional 1 ml of Ni-NTA resin. After thorough washsteps using the wash buffer (50 mM phosphate, 300 mM NaCl, pH 7.0) with0-10 mM imidazole, the PEP was eluted with 150 mM imidazole, 50 mMphosphate, 300 mM NaCl, pH 8. FM PEP was further purified on a FPLCsystem (Amersham Pharmacia, NJ) through a HiTrap-SP cation exchangecolumn. Prior to application on the HiTrap-SP column, the protein wasexchanged into 20 mM phosphate buffer (pH 7). Following injection, PEPwas eluted with a salt gradient from 20 mM phosphate, pH 7 (buffer A) to20 mM phosphate, 500 mM NaCl, pH 7 (buffer B) at a flow rate of 1ml/min. MX PEP, a cytosolic protein, was initially purified from awhole-cell lysate via Ni-NTA affinity chromatography (as detailedabove). The protein was further purified on a Superdex 200 gelfiltration column (Amersham) with an isocratic gradient of 20 mM HEPES,2 mM DTT, pH 7.0 at 1 ml/min.

Activity Assays. Post-proline cleavage activity was measured usingZ-Gly-Pro-p-nitroanilide and Succinyl-Ala-Pro-p-nitroanilide (Bachem,CA). Z-Gly-Pro-pNA was dissolved in a PBS:water:dioxane (8:1.2:0.8)assay mixture. The concentration of Z-Gly-Pro-pNA was varied from100-600 μM. Although the substrate Z-Gly-Pro-pNA was effective indetecting enzyme activity, its insolubility at higher concentrationsprecluded kinetic measurements under substrate-saturated conditions. Incontrast, Succinyl-Ala-Pro-pNA, had the advantage of high watersolubility at all pH values tested, and was therefore a preferredsubstrate for kinetic studies. Hydrolysis of Suc-Ala-Pro-pNA by FM, SCand MX PEPs was monitored in a reaction mixture (300 μA consisting of 30μA of 10×PBS buffer, a final concentration of 0.01-0.02 μM enzyme, andSuc-Ala-Pro-pNA (5 mM stock) at final concentrations ranging between 100μM to 4 mM. The release of the p-nitroanilide was spectrophotometricallydetected at a wavelength of 410 nm. The initial velocity of the reactionwas determined by the increase in absorbance at 410 nm, which was usedto calculate Km and Kcat according to the Michaelis-Menten relationship.For measurement of the influence of pH on the enzyme activity, a seriesof pH buffer solutions were prepared using citric acid and disodiumphosphate for pH values from 3.0 to 6.0, and sodium phosphates for pHvalues from 7.0 to 8.0. Reaction mixtures (300 μA consisted of 30 μl of10× pH buffer, final concentration of 0.01 μM enzyme, andSuc-Ala-Pro-pNA to final concentrations between 100 μM to 4 mM.

pH Stability. The ability to retain enzyme activity after exposure toacidic environments was determined. Hydrochloric acid solutions (10 μAat pH values ranging from 1.5 to 4.0 were mixed with 1 μA of enzyme for10-20 min. The acidic mixtures were then neutralized with 40 μA of10×PBS solution, 60 μA of 5 mM substrate to a final volume of 300 μl.The recovered enzyme activity was measured spectrophotometrically andcompared with non-acid treated controls under identical conditions.

Gastric and Pancreatic Protease Stability. In a 96-well U-bottomedplate, 5 μL of 2× reaction buffer (40 mM Na₂HPO₄, pH=6.5 for pancreaticenzymes or 20 mM HCl for pepsin) was placed, and 1 μL of the degradingenzyme (either 1 mg/ml pepsin or a cocktail of 1 mg/ml trypsin, 1 mg/mlchymotrypsin, 0.2 mg/ml elastase and 0.2 mg/ml carboxypeptidase A)followed by 4 μL of PEP (5-10 U/ml) were added. The plate was incubatedat 37° C. for various times (e.g. 0, 5, 10, 20 and 30 min), with 190 μLof PEP substrate solution (2 μl Z-Gly-Pro-p-nitroanilide (16.8 mg/ml indioxane) 14 μl dioxane, 24 μl water, 150 μl 10 mM PBS buffer, pH=7.5)added to each well. Absorption was measured at 410 nm for 1 to 2 minevery 10 s to assay residual activity. Each buffer also contained 5mg/ml gluten. Untreated gluten was used for pepsin, whereas glutenpreviously proteolyzed with pepsin (0.01 M HCl, pH=2.0, 1:50 w/w, 2 h,37° C.) was used for all other enzymes. Wells containing acid (pH=2.0)were neutralized by addition of 10 μL 0.1 M NaOH before addition of thePEP substrate. Enzyme activities are expressed as a percentage of themaximum activity, typically observed at the zero time point.

Substrate Specificity. In addition to the reference substrates above,enzyme specificity was also evaluated using two immunogenic peptidesderived from the sequence of γ-gliadin proteins in gluten. Both peptideswere synthesized using solid-phase peptide synthesis. The peptide (SEQID NO:4) PQPQLPYPQPQLP contains the immunodominant γII-epitope, and isresistant to proteolysis by pepsin or any pancreatic enzyme. PEPspecificity toward this substrate was assessed in a competitive assay inwhich 100 μM (SEQ ID NO:4) PQPQLPYPQPQLP and 100 μM Suc-Ala-Pro-pNA weremixed and reacted with 0.02 μM PEP at 25° C. The initial velocity ofSuc-Ala-Pro-pNA cleavage was measured spectrophotometrically, whereasthe initial velocity of (SEQ ID NO:4) PQPQLPYPQPQLP hydrolysis wasdetermined via HPLC. The apparent specificity, _(kca)t/_(K)M, for thehydrolysis of (SEQ ID NO:4) PQPQLPYPQPQLP could be determined based onthe known _(kca)t/_(KM) of the enzyme for Suc-Ala-Pro-pNA and theobserved reaction rates of the two substrates. In addition toPQPQLPYPQPQLP, PEP specificity for the more complex but physiologicallyrelevant peptide (SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF(33-mer) was also assessed. Proteolysis reactions were performed at 37°C. in PBS buffer with 5-100 μM peptide and 0.1 μM PEP for time periodsof 1 min-4 hrs.

The decrease in substrate concentration as well as concomitantintermediate and product build-up were monitored with HPLC analysis.RP-HPLC was performed on a system consisting of Beckman or RaininDynamax SD-200, a Varian 340 UV detector set at 215 nm and 280 nm.Solvent A was H2O with 0.1% TFA and solvent B was acetonitrile with 0.1%TFA; gradient used: 0-5% B in 0-15 min, 5-30% B in 15-30′, 30-100% B in30-35 min, 100% B for 5′; flow 1 ml/min; separation was performed on a4.6×150 mm reverse phase C-18 column (Vydac, Hesperia, Calif., USA).Samples were centrifuged for 10 min at 13,400 g, prior to injection of10-100 μl. Both (SEQ ID NO:4) PQPQLPYPQPQLP as well as the 33-mer havemultiple post-proline endoproteolytic sites. Thus, multiple peptidesaccumulate during the course of the reaction, some of which aresecondary PEP substrates in themselves. Electrospray-Ion Trap-MS-MScoupled with a UV-HPLC (LCQ Classic/Surveyor, ThermoFinnigan, CA) wasused to identify the preferred cleavage sites in (SEQ ID NO:4)PQPQLPYPQPQLP and the 33-mer.

For further evaluation of the proteolysis of the 33-mer and (SEQ IDNO:4) PQPQLPYPQPQLP in the appropriate physiological environment, gluten(30 g/L) was suspended in 0.01 M HCl (pH=2.0) and incubated in thepresence of pepsin (600 mg/L) for 2 h at 37° C. The resulting solutionwas neutralized using 10 M NaOH and diluted to 10 g/L in a phosphatebuffer (40 mM, pH 6.5). 25 μA of this suspension were then supplementedwith the 33-mer (0.1 mg/ml), (SEQ ID NO:4) PQPQLPYPQPQLP (0.08 mM),trypsin (0.1 mg/ml), chymotrypsin (0.1 mg/ml), elastase (0.02 mg/ml),carboxypeptidase A (0.02 mg/ml). Prolyl endopeptidase (FM or MX; 1×: 500mU/ml; 5×: 2.5 U/ml; 10×: 5 U/ml) and rat intestinal brush bordersurface membranes (BB, 1×: 40 mU/ml, 2×: 80 mU/ml, DPP IV activity) wereadded to a total volume of 150 μl. The mixture was incubated at 37° C.and 25 μA aliquots were taken at 0, 5, 10, 30 and 60 min and immediatelyheat deactivated.

To examine the chain length specificity of individual PEPs, we performedcompetitive reactions containing both gluten-derived peptides, subjectedthe reaction mixture to RP-HPLC, and monitored the disappearance of eachsubstrate was monitored as a function of time. The peak areas of the33-mer (32.5 min) and (SEQ ID NO:4) PQPQLPYPQPQLP (27.5 min) wereintegrated.

In Vivo Endopeptidase Activity. An adult (female or male) rat wasanesthetized and maintained at 36-37° C. during the entire surgicalprocedure. The peritoneal cavity was opened, and a small incision wasmade at the beginning and the end of a 15-20 cm jejunum segment.Polyethylene catheters were inserted and secured into the two ends. Theinput catheter was connected with a pump-driven syringe filled with asolution. The jejunum segment was perfused initially with PBS buffer toremove any residual debris at a flow rate of 0.4 ml/min. Purifiedpeptide solutions (peptide concentration ranges from 25-100 μM) werethen perfused at 0.4 ml/min through the jejunum segment with a 10-40 minresidence time. In the case of a co-perfusion, the input catheter isconnected with two simultaneous syringes, one with a peptide solutionand the other with the prolyl endopeptidase solution (concentrationranges from 50-500 μU/μl). Fluid from the output catheter was collectedinto small centrifuge tubes in dry ice for subsequent analysis. Thecollected digestive products were analyzed by HPLC on a C18 column.

Results

PEP Protein Expression. FM and SC PEPs have their own signal sequences,and were therefore expressed as secreted, soluble enzymes in theperiplasmic space of E. coli. A simple freeze-thaw lysis procedure ledto recovery of periplasmic protein without significant contamination bycytoplasmic proteins. In contrast, the MX PEP lacks a native signalsequence, and was therefore expressed as a cytoplasmic protein. PEP waspurified from each lysate by Ni-NTA affinity purification, followed by asecond chromatographic step. The yields of active FM, SC and MX PEPswere 1 mg/L, 60 mg/L and 30 mg/L, respectively. The purity of thevarious PEPs was determined by SDS-PAGE to be >90%.

Kinetic Analysis with Reference Substrates. The activity of each PEP wasinitially evaluated using the standard chromogenic substratesuccinyl-Ala-Pro-pNA. Release of the p-nitroaniline was detected at 410nm, and kinetic data was fitted to the Michaelis-Menten relationship.Succinyl-Ala-Pro-pNA was selected as a reference substrate instead ofthe more commonly used Z-Gly-Pro-pNA due to the low solubility of thelatter substrate, which necessitated use of co-solvents. The calculated_(kcat) and _(KM) values of FM, MX and SC PEPs for succinyl-Ala-Pro-pNAare tabulated (Table 3). While these enzymes all exhibited comparablelevel activity to that of a serine protease, MX PEP has a higherspecificity than the FM PEP, whereas SC PEP has an intermediate level ofspecificity (Table 4). The higher specificity of MX can be attributedmainly to its higher affinity for the substrate, as reflected in theK_(M).

TABLE 3 Kinetic parameters for Succinyl-Ala-Pro-p-nitroanilidehydrolysis by FM PEP, MX PEP and SC PEP. K_(cat) (s⁻¹) K_(M) (mM)K_(cat)/K_(M) (mM⁻¹/s⁻¹) FM PEP 33 0.91 37 MX PEP 51 0.35 146 SC PEP 1442.1 67

TABLE 4 Specificity of FM PEP, MX PEP and SC PEP for the immunogenicgliadin peptide (SEQ ID NO: 4) PQPQLPYPQPQLP. K_(cat)/K_(M) (mM⁻¹/s⁻¹)FM PEP 178 MX PEP 548 SC PEP 492

Enzyme Activity vs. pH. The luminal environment of the duodenum isapproximately at pH 6. Therefore, a therapeutically useful PEP mustretain high specific activity at that pH. The steady state turnoverrate, kcat, of each PEP was titrated in various pH conditions using100-4000 μM succinyl-Ala-Pro-pNA, shown in FIG. 8. Both FM PEP and MXPEP exhibited active site pKa around pH 6, indicating optimal activityin the pH 6-8 range. The diminished activity of both enzymes at pH 5 isconsistent with the well-established role of a histidine residue as thegeneral base in the serine protease catalytic triad, but alternativelyit may indicate a change from the active enzyme conformation to aninactive state. Such conformational changes have been implicated in thecatalytic cycle of the structurally characterized porcine brain PEP.Interestingly, the SC PEP, which has the broadest pH profile, shows amarked increase in maximum velocity under weakly basic conditions.

PEP Stability. Although orally administered therapeutic proteins can beformulated to protect them from the acidic and proteolytic environmentof the stomach, intrinsic acid stability of a PEP is likely to be adesirable characteristic in its use as a therapeutic agent for CeliacSprue. We therefore evaluated the extent to which the activity of eachPEP remains intact after 10 min of incubation at selected pH valuesbetween 1.6 and 3.9. Within this pH range, the FM PEP retained 50-70% ofits original activity; the MX PEP retained 70-90% activity; and the SCPEP retained 30-80% activity. Thus, although all PEPs appear to bemoderately acid-stable, the MX PEP is most versatile. Since therapeuticefficacy would require a PEP to act upon gluten in conjunction withpancreatic proteases that are secreted into the duodenum, the resistanceof FM PEP and MX PEP toward both gastric and pancreatic enzymes wasevaluated. For this we pre-incubated the enzymes with physiologicalquantities of either pepsin (at pH 2) or a cocktail comprising oftrypsin, chymotrypsin, elastase and carboxypeptidase A (at pH 6.5). Ascan be seen in FIG. 9, both FM and MX PEP were highly susceptible topepsin catalyzed proteolysis, whereas they appear to be remarkablystable to destruction in the presence of physiological quantities of thepancreatic enzymes.

Kinetic analysis using PQPQLPYPQPQLP as a substrate. The immunogenicpeptide PQPQLPYPQPQLP is a recurring sequence in γ-gliadins, and isresistant to proteolysis by gastric and pancreatic proteases. It is alsohighly resistant to digestion by intestinal brush border peptidases,with only dipeptidyl carboxypeptidase I (DCP1) able to act upon it.Treatment of this peptide with PEP results in cleavage at internalproline residues, which in turn generates new recognition sites forbrush border aminopeptidases. Thus, (SEQ ID NO:4) PQPQLPYPQPQLPrepresents a good test substrate to probe PEP specificity.

The k_(cat)/K_(M) values of each PEP were determined in an assay mixturecontaining (SEQ ID NO:4) PQPQLPYPQPQLP as well as Suc-Ala-Pro-pNA as acompeting substrate. The rates of disappearance of both substrates weredetermined by independent detection methods. The initial rate ofdisappearance of (SEQ ID NO:4) PQPQLPYPQPQLP was measured by HPLC,whereas the rate of consumption of Suc-Ala-Pro-pNA was measuredspectrophotometrically. Both FM and MX PEP had a 5-fold higherspecificity for the gluten peptide as compared to the chromogenicsubstrate, whereas the SC PEP showed a 7-fold increase in specificityfor the gluten peptide (Table 4). This increase in specificity suggeststhat longer peptides may provide additional anchors at the catalyticsite, a hypothesis that is consistent with the observation thatAla-Pro-pNA (which lacks an N-terminal succinyl group or a carboxybenzylgroup) did not react with any of the PEPs.

To analyze the regiospecificity of hydrolysis of (SEQ ID NO:4)PQPQLPYPQPQLP by individual PEPs, samples corresponding to early timepoints were further analyzed by LC/MS/MS. The results, shown in FIG.10A-10D, reveal that each PEP has unique subsite preferences. While thepreferred site of cleavage by FM PEP was at the (SEQ ID NO:4)PQPQLPPYP|QPQLP position, MX PEP preferentially cleaved the same peptideat the (SEQ ID NO:4) PQPQLP|YPQPQLP position. SC had comparablepreference for either site of cleavage. All enzymes preferentiallycleaved the peptide at a proline located near the middle of thesequence, highlighting their functional difference from prolyl-specificexopeptidases such as DPP IV.

Chain Length Tolerance and Selectivity. It has been suggested thatprolyl endopeptidases from the serine protease family are limited withregard to chain lengths of potential substrates. To test this hypothesisin the context of the three bacterial PEPs studied here, we comparedtheir hydrolytic activities against a physiologically relevant 33-merpeptide sequence from wheat gliadin, (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLP YPQPQPF (FIG. 11A). The FM PEP (0.1 μM) wasable to hydrolyze 10 μM of the 33-mer in about 2-3 minutes, whereas theSC PEP required >1 hr to reach a comparable endpoint. Based on initialrates, the FM PEP was estimated to act 5-fold faster on the 33-mer thanthe MX PEP, and >20 fold faster than the SC PEP. Thus, the SC PEPappears to have a severe chain length restriction for long peptidesubstrates.

The intermediates and products from hydrolysis of the 33-mer by the FMand MX PEPs were analyzed by LC/MS/MS (FIG. 11B-C). Several features arenoteworthy. First, even at relatively early time-points, the digestiveproducts of the MX PEP were predominantly small fragments, whereas FMPEP digestion yielded a significant pool of long intermediates such as(SEQ ID NO:12, aa 1-19) LQLQPFPQPQLPYPQPQLP, (SEQ ID NO:12, aa 1-14),LQLQPFPQPQLPYP and (SEQ ID NO:12, aa 1-12), LQLQPFPQPQLP. Thus, althoughboth PEPs are able to effectively proteolyze the 33-mer, they havedistinct hydrolytic patterns on this complex substrate. In particular,either the MX PEP appears to be processive (i.e. for each 33-mersubstrate molecule, it sequentially cleaves all the preferred sites inthe chain prior to release), or alternatively the enzyme has a strongbias toward shorter chain substrates. It could also be noted that theC-terminal fragments generated by the two enzymes are different (QPQPFfor the FM PEP, and YPQPQPF for the MX PEP). This finding is consistentwith observed sub-site preference in the case of (SEQ ID NO:4)PQPQLPYPQPQLP digestion.

To directly investigate chain length selectivity of the three enzymes,we co-incubated (SEQ ID NO:4) PQPQLPYPQPQLP and (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF with each PEP (FIG. 13A-C) Both the SCPEP and the MX PEP showed a clear preference for the 13-mer peptide,whereas the FM PEP showed comparable selectivity for both peptides.

To further evaluate the substrate preferences, (SEQ ID NO:4)PQPQPLPYPQPQLP and the 33-mer were mixed with pepsin-treated gluten, andallowed to react with pancreatic enzymes in the presence of BBM andeither FM PEP or MX PEP. As seen in the HPLC traces (FIG. 13A-B), the33-mer had the longest retention time, whereas (SEQ ID NO:4)PQPQLPYPQPQLP and other medium-length gluten peptides eluted earlier.Here too the FM PEP proteolyzed (SEQ ID NO:4) PQPQLPYPQPQLP, the 33-merand other gluten peptides at comparable rates (FIG. 13A). In the MX PEPdigestion, PQPQLPYPQPQLP and other smaller peptides were rapidly brokendown (in 10 minutes), whereas hydrolysis of the 33-mer occurred at aslower rate (FIG. 13B).

In Vivo Hydrolysis. To validate the implications of the abovebiochemical observations for peptide digestion in the intact smallintestine, each PEP was co-perfused in the rat jejunum with the 33-merpeptide substrate, and the effluent collected at a distance of 15-20 cmfrom the point of perfusion was analyzed. In this live animal model, theimpact of concerted action of the perfused (luminal) PEP and the brushborder (surface) peptidases is assessed. As shown by the in vitroresults above, while the BBM enzymes were insufficient to process the33-mer, FM PEP promoted more complete breakdown of the 33-mer than boththe MX and the SC PEP (FIG. 14). Within a PEP dose range of 50-500μU/μl, the extent of 33-mer hydrolysis increased with increasing PEPdose, demonstrating that higher doses of PEP could accelerate glutenbreakdown in the mammalian gut.

In light of recent findings that related the strong antigenicity ofgliadin peptides to their exceptional digestive resistance, prolylendopeptidases were identified as a potentially interesting family ofenzymes for oral Celiac Sprue therapy. Understanding the enzymologicalproperties of these enzymes is an essential prerequisite for such use.In the above study, prolyl endopeptidases from three bacterial sourceswere selected and expressed in E. coli as recombinant proteins, and weresubsequently purified and characterized. Two of these enzymes (from F.meningosepticum and S. capsulate) have been reported earlier, whereasthe third enzyme (from M. xanthus) represents a new member of the prolylendopeptidase family.

In order to examine the endoproteolytic properties of these enzymes, itis important to utilize peptide substrates with internal cleavage sites.Although model substrates such as Z-Gly-Pro-pNA or Suc-Ala-Pro-pNA havebeen frequently used to identify and characterize polyl endopeptidases,these substrates alone do not provide adequate insight to differentiateendopeptidases from each other or from proline-specific aminopeptidases(such as dipeptidyl peptidase IV (DPP IV)). In the context of CeliacSprue, two peptides ((SEQ ID NO:4) PQPQLPYPQPQLP and (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF) have been recognized as useful probesfor studying the fundamental properties of prolyl endopeptidases, aswell as for their potential for detoxifying gluten. The peptide (SEQ IDNO:4) PQPQLPYPQPQLP contains an epitope found in γ-gliadins that hasbeen shown to play an immunodominant role in the T cell mediatedresponse to gluten in the Celiac gut. It cannot be cleaved by anygastric or pancreatic proteases and is also highly resistant todigestion by intestinal brush border membrane (BBM) peptidases, withonly dipeptidyl carboxypeptidase I able to act upon it at a very limitedrate. Thus, the efficiency of intestinal metabolism of this peptide canbe expected to improve in the presence of an exogenous prolylendopeptidase, as has been verified in this study. Treatment of thispeptide with PEP results in cleavage at an internal proline residue,which in turn generates a new recognition site for brush borderaminopeptidases. Thus, (SEQ ID NO:4) PQPQLPYPQPQLP represents a goodprobe for PEP specificity.

The 33-mer gliadin peptide (SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF was selected as a complementary probe for these studies, becauseit is a stable, physiologically derived product of gastric andpancreatic digestion of γ-gliadin, and strongly stimulates proliferationof gluten-reactive T cells from virtually all Celiac Sprue patientstested thus far. Therefore, endoproteolytic breakdown of this 33-merpeptide represents an especially challenging goal for an exogenous PEP.Like most other antigenic gluten peptides, the 33-mer contains multipleproline residues, and can be expected to present more than one cleavagesite to a PEP. At the same time its multivalent character suggests thatPEP action alone is unlikely to eliminate all residual antigenicity ofthis peptide. Consequently, combined action of a PEP and the endogenouspeptidases of the intestinal brush border membrane is required forimmunological neutralization and dietary assimilation of this longproline-rich peptide.

Our investigations into the molecular recognition features of threebacterial PEPs for two gliadin peptides have revealed at least twointeresting and potentially important characteristics of these enzymes.First, although all three PEPs tested here exhibited high specificactivity against reference chromogenic substrates (Table 3), they showedremarkable differences in chain length specificity (FIG. 10A-C). WhereasSC PEP and MX PEP had higher specificity for (SEQ ID NO:4) PQPQLPYPQPQLPthan FM PEP (Table 4), the reverse was true for the longer 33-mergliadin peptide (FIG. 11A), especially in the case of the SC PEP, whichhad extremely poor activity against the 33-mer.

Structural and biochemical analysis led to the proposal that theactivity of PEPs is limited to substrates containing fewer than 30 aminoacid residues. In that light the good activity of MX PEP and especiallyFM PEP against the 33-mer peptide is surprising. The broad chain lengthtolerance of FM PEP is vividly demonstrated in competitive in vitro andin vivo assays, where FM PEP was able to process longer and shortersubstrates at comparable rates. Second, sequence analysis of the majorproteolytic products derived from both gliadin substrates demonstratedthat the PEP's had distinct sub-site specificity as well asregiospecificity in the context of the longer repetitive sequence. Forexample, the FM PEP preferentially cleaved at (SEQ ID NO:4)PQPQLPYP|QPQLP, whereas the MX PEP preferred the (SEQ ID NO:4)PQPQLP|YPQPQLP site, and the SC PEP had comparable activity towardeither site.

Similarly, sequence analysis of initial hydrolytic products of the33-mer peptide underscored regiochemical differences between FM PEP andMX PEP. Whereas MX PEP treatment generated fragments mostly of 4-5residues (presumably processed sequentially from both termini), FM PEPyielded longer intermediates (presumably as a result of a preferentialcleavage near the center of the peptide). Thus, the active sites ofthese enzymes are clearly different, which in turn has potentialimplications for the use of these enzymes detoxifying dietary gluten fora Celiac Sprue patient.

In addition to analyzing substrate specificity, we have alsoinvestigated other therapeutically relevant properties of our set ofthree PEPs. They include pH dependence of enzyme activity, acidtolerance of the protein, and resistance toward inactivation by gastric,pancreatic and intestinal proteases/peptidases. All enzymes have a pHactivity profile that is well matched to the mildly acidic environmentof the upper small intestine (pH 6-6.5) (FIG. 3). They also appear to bemoderately stable toward acid exposure as well as pancreatic protease(but not pepsin) action, with the MX PEP being the most stable (FIG. 8).The enzymes also retain activity in the intact small intestinal lumen ofa rat, indicative of their stability toward both intestinal secretionsas well as brush border membrane peptidases (FIG. 14). Finally, theexpression levels of these enzymes vary significantly in recombinant E.coli. Specifically, in comparison to the FM PEP, the expression levelsof SC and MX PEPs were substantially superior.

The porcine brain PEP has a didomain architecture, including an unusualβ-propeller domain that appears to regulate proteolysis. Pairwisesequence alignments between this structurally characterized PEP and FM,MX and SC PEP reveal 39% (49%), 36% (45%) and 40% (48%) identity(similarity), respectively. These alignments also suggest that thebacterial PEPs are comprised of a catalytic and a β-propeller domain.Since their active sites are predicted to lie near the interface betweenthe two domains, mutagenesis at the inter-domain interface could alterprotein dynamics and in turn affect substrate tolerance and specificity.

The above results provide a basis for protein engineering efforts of PEPenzymes. This family of serine proteases includes numerous otherputative homologs whose cDNAs have been sequenced but whose geneproducts remain to be characterized. In light of the favorableproperties of the MX PEP, which was expressed and characterized for thefirst time as part of this study, it will be useful to screen additionalwild-type enzymes.

Example 4 Heterologous Expression of PEP in Lactobacilli

In one embodiment of the present invention, a Celiac Sprue patient isprovided with a recombinant organism modified to express a PEP of theinvention. The recombinant organism is selected from those organismsthat can colonize the intestinal mucosa without detriment to thepatient, thereby providing an endogenous source of PEP to the patient.As one example, Lactobacilli such as L. casei and L. plantarium cancolonize the intestinal mucosa and secrete PEP enzymes locally. Giventheir widespread use in food processing, they can also be used as anefficient source of PEP for industrial (to treat foodstuffs) and medical(to prepare PEP for pharmaceutical formulation) use. PEPs can beexpressed in such lactobacilli using standard recombinant DNAtechnologies. For example, Shaw et al. (Shaw, D M, Gaerthe, B; Leer, RJ, Van der Stap, JGMM, Smittenaar, C.; Den Bak-Glashouwer, Heijne, M J,Thole, J E R, Tielen F J, Pouwels, P H, Havenith, CEG (2000) Immunology100, 510-518) have engineered Lactobacilli species to expressintracellular and surface-bound tetanus toxin. The intact PEP genes(including leader sequences for efficient bacterial secretion) can becloned into shuttle expression vectors such as pLP401 or pLP503 undercontrol of the (regulatable) amylase promoter or (constitutive) lactatedehydrogenase promoter, respectively. Alternatively, recombinant foodgrade Lactobacilli strains can be generated by site specificrecombination technology (e.g. see. Martin M C, Alonso, J C, Suarez J E,and Alvarez M A Appl. Env. Microbiol. 66, 2599-2604, 2000). Standardcultivation conditions are used for Lactobacilli fermentation, such asthose described by Martin et al.

Example 5 Heterologous Expression of PEP in Yeasts

Both naturally occurring and recombinant cells and organisms can be usedto produce the glutenases useful in practice of the present invention.Preferred glutenases and producing cells include those from organismsknown to be Generally Regarded as Safe, such as Flavobacterium,Aeromonas, Sphingomonas, Lactobacillus, Aspergillus, Xanthomonas,Pyrococcus, Bacillus and Streptomyces. Extracellular glutenase enzymesmay be obtained from microorganisms such as Aspergillus oryzae andLactobacillus casei. Preferred cells include those that are already usedin the preparation of foodstuffs but have been modified to express aglutenase useful in the practice of the present invention. As oneexample, yeast strains such as Saccharomyces cerevisiae are useful forhigh level expression of secreted heterologous proteins. Genes encodingany of the PEPs described above (mature protein only) can be cloned inexpression plasmids designed for optimal production of secretedproteins. An example of such a heterologous expression strategy isdescribed in Parekh, R. N. and Wittrup, K. D. (Biotechnol. Prog. 13,117-122, 1997). Either self-replicating (e.g. 2 micron) or integrating(e.g. pAUR101) vectors can be used. The GAL1-10 promoter is an exampleof an inducible promoter, whereas the ADH2 promoter is an example of aconstitutive promoter. The cDNA encoding the mature PEP is fuseddownstream of a leader sequence containing a synthetic pre-pro regionthat includes a signal cleavage site and a Kex2p cleavage site. S.cerevisiae BJ5464 can be used as a host for production of the peptidase.Shake-flask fermentation conditions are described by Parekh and Wittrupin the above-cited reference. Alternatively, high cell density fed-batchcultures can be used for large scale production of the peptidases; arepresentative procedure for this purpose is described in Calado, C. R.C, Mannesse, M., Egmond, M., Cabral, J. M. S, and Fonseca, L. P.(Biotechnol. Bioeng. 78, 692-698, 2002).

Example 6 Enteric Capsule Formulation of Prolyl Endopeptidase

Gelatin capsules are filled with 100 mg prolyl endopeptidase and 10 mgof silicon dioxide. The capsules are enterically coated with Eudragitpolymer and put in a vacuum chamber for 72 hours. The capsules are thenheld at a range of temperature of 10° C. to 37° C. and a controlledhumidity level of 35-40%.

Example 7 Studies of Enteric Capsule Formulation of Prolyl Endopeptidase

A study is conducted where patients with Celiac Sprue are enrolled in atwo week-long study. Gelatin capsules containing 90% prolylendopeptidase mixed with 10% silicon dioxide are used. The capsules arehand-filled with the mixture, banded, and coated with a 10% Suretericenteric coating (a polymer of polyvinylacetatephthalate developed by theCanadian subsidiary of Merck & Company). Samples are acid-tested byexposing the coating to 1N HCL for one hour in order to simulate theacid environment of the stomach. The capsules are then put in a vacuumchamber for 72 hours.

Two 100 mg capsules are administered to each patient prior to each meal.The patients are instructed to eat all kinds of food without abstainingfrom those that were known to cause distress, e.g., bloating, diarrhea,and cramps.

Example 8 Enteric Pill Formulation of Prolyl Endopeptidase

400 mg of L-tartaric acid and 40 mg of polyethylene glycol-hydrogenatedcastor oil (HCO-60) are dissolved in 5 ml of methanol. This solution isplaced in a mortar previously warmed to 30° C. To the solution is added100 mg of prolyl endopeptidase. Immediately after the addition of PEP,the mixture is stirred with a pestle under a hot air current (40° C.)and then placed in a desiccator under vacuum overnight to remove thesolvent. The resulting solid-mass is pulverized with a pestle andkneaded with 30 mg of sodium bicarbonate and a small amount of 70%ethanol. The mixture is then divided and shaped into pills of about 2 mmsize and thoroughly dried. The dried pills are given a coating ofhydroxypropylmethylcellulose phthalate (HP-55) to obtain an entericformulation.

Example 9 Endoprotease Activity

The gene for an endoprotease (EPB2; PubMed accession number U19384, nt94-1963) from barley (Hordeum vulgare subsp. vulgare) was subcloned intoa pET28b (Invitrogen) vector using BamH1 and EcoR1 insertion sites; theresulting plasmid was designated pMTB1. An inactive 43 kDa proproteinform of EPB2 was expressed from pMTB1 in the cytoplasm of BL21 E. colicells. The proprotein was solubilized from the inclusion bodies using 7M urea. The solubilized protein was purified on a Ni-NTA column.Auto-activation of proEPB2 to its mature, active form was achieved byaddition of citrate-phosphate buffer, pH 3 (prepared by mixing 0.1 Msodium citrate and 0.2 M sodium phosphate). Under such acidicconditions, proEPB2 converts rapidly into a mature form with a molecularweight of 30 kDa (FIG. 2). By 72 hours, mature EPB2 undergoes autolysis.N-terminal sequencing yielded an N-terminal sequence beginning withVSDLP.

Under acidic conditions, the mature form of EPB2 efficiently digestspurified α2-gliadin, a source of peptides that are immunogenic to peoplewho suffer from Celiac Sprue. The cysteine proteinase inhibitor,leupeptin, inhibits this activity, confirming its mechanism as acysteine protease. The pH optimum of proEPB2 activation and α2-gliadindigestion is 2.4-3.5, which can therefore provide a treatment for CeliacSprue consisting of oral administration of proEPB2.

Example 10 Formulation and Efficacy Analysis of M. xanthus PEP

Lyophilization of M. xanthus PEP was performed as follows. The PEP waspurified as described in Example 3, and concentrated to an initialconcentration of 7.7 mg/ml by Tangential-Flow Filtration (TFF) using a10K MWCO Pellicon difiltration membrane (Millipore, PLCGC10, 50 cm, Cat.No. PXC010C50). TFF (using a LabScale TFF from Millipore, Cat. No.29751) was performed for approximately 12 hours (pressure of 50 psi(retentate)/30 psi (permeant)), with periodic addition to the reservoirof 50 mM Sodium Phosphate, 3% Sucrose pH 7.5. Thereafter, PEG-4000 wasadded with a target concentration of 1%. The final protein concentrationwas 70-100 mg/ml. This material was centrifuged, then lyophilized. Thelyophilization was performed in square petri dishes (Falcon Cat. No.35-1112) in a DuraStop lyophilizer using parameters outlined in theTable below. Typically, 0.7-0.85 mg PEP was present per mg oflyophilized material. No loss of specific activity of the PEP wasobserved upon lyophilization.

Step Temperature Pressure Duration Ramp Rate Freezing 1 −50° C.Atmospheric 2 hrs 0.3° C./minute Annealing −35° C. Atmospheric 3 hrs0.3° C./minute Freezing 2 −50° C. Atmospheric 2 hrs 0.3° C./minute 1°Drying −20° C. 100 mTorr 16.9 hrs 0.5° C./minute 2° Drying +25° C. 100mTorr 8.0 hrs 0.2° C./minute *P. Temp. = Avg. Product Temp. at end ofstep. **1° = Primary Drying. ***2° = Secondary Drying

Blending for the M. xanthus PEP was performed as follows. Lyophilizedcakes were pulverized to a light powder. All samples were weighed forrecovery and stored in sealed 50 mL conical vials at 4° C. A blend wasprepared as shown below. The excipients were selected to provide properflow and disintegration properties for the blended mixture.

Order of Addition Excipient Percentage 1 Lyophilized enzyme 63%cake/powder 2 Calcium Silicate  2% 3 Talc  5% 4 Crospovidone  5% 5Avicel 25%

The lyophilized enzyme and excipients were blended in a V-blender forseveral hours. The material was then used to make enteric-coatedcapsules or tablets. 100-150 mg M. xanthus PEP could be loaded into asingle hard gelatin capsule, size 00 (Capsugel). Alternatively, Vcapvegetable capsules (size 00, Capsugel) can also be used with no impacton enzyme activity.

For enteric coating of the capsules, an enteric coating solution wasprepared as shown below:

Order of Addition Excipient Amount added 1 RODI water 49.5 mL 2 Talc 8.1g 3 Eudragit L50 D-55 111.0 mL 4 Triethyl Citrate 1.62 mL

The enteric coating was mixed vigorously in a beaker on a stir plate.The solution was then decanted into a spray bottle. Rat capsules werecarefully spread on paper towels in groups of 20 and the enteric coatingsolution sprayed onto the capsules. Warm air was used to partially drythe capsules before moving them to a dry paper towel where theyair-dried for 30 minutes before the next coat was applied. A total of 3coats were applied in order to cover all sides of the capsules. Thesewere air dried several hours before being transferred to a storagecontainer. Although some activity of the PEP is lost as a result ofenteric coating, a substantial fraction of the activity is retained, andis stable for at least 1 month at 4 C storage.

An alternative method to formulate the enzyme for intestinal delivery isas an enteric-coated tablet. Tablets have the advantage of more rapiddissolution in the weakly acidic environment of the upper smallintestine. Another advantage of the tablet formulation is that moreenzyme can be compacted into a smaller volume than for a capsule. Theirprimary liability is that proteins frequently denature under highpressures. In a method of tablet preparation of M. xanthus PEP, the samelyophilized blend as above was used. Tablets were prepared at a punchstrength of 3000 psi held for 15 seconds. No activity was lost in theprocess, demonstrating the feasibility of tablet formulations of thisenzyme.

To test the efficacy of the enteric-coated oral capsule formulationdescribed above, two types of tests were performed. In vitro dissolutiontests were performed on a Hanson SR8-Plus Dissolution Tester usingSimulated Gastric Fluid (SGF; 2 g/L NaCl, pH 1.2, adjusted using 6 NHCl) and Simulated Intestinal Fluid (SIF; 6 g/L monobasic potassiumphosphate with or without 10 g/L pancreatin, pH 6.8, adjusted using 5 NNaOH). Enteric coated capsules were first tested for resistance todissolution in SGF for up to 2 h at 37° C. No protein release was noted.Subsequently the capsules were subjected to similar dissolution tests inSIF at 37° C. A substantial fraction of the encapsulated material wasreleased in 15 min. By 30 min the material had been completely released.

In vivo tests of the capsules were performed in rats using smaller hardgelatin capsules (Size 9 capsules, Torpac). Approximately 16 mg of thelyophilized formulation blend was encapsulated in each enteric-coatedcapsule, corresponding to −7 mg PEP. Rats fasted overnight wereadministered via oral gavage one PEP or placebo capsule along with ameasured quantity (300 mg gluten/kg body weight) of gluten syrupprepared as follows. 300 g commercially available wheat gluten flour(Bob's Red Mill, Milwaukie Oreg.) was added to 10 L of a 0.01 M HClsolution to achieve a pH of 2.0. Pepsin (6.0 g, American Laboratories)was added. After incubation at 37° C. for 1 h, the pH was adjusted to2.0 by addition of 35 ml 1M HCl. After maintenance for an additional 2 hat 37° C., the solution was neutralized by addition of 35 g of Na₂HPO₄,and the pH was adjusted to 7.9 with 10 M NaOH (32.5 ml).Trypsin/Chymotrypsin powder (3.75 g) (Enzyme Development Corp; 1000USP/mg in trypsin, 1000 USP/mg in chymotrypsin) was then added, thereaction maintained at 37° C. for 2 hours, pH 7.9 (pH re-adjustment to7.9 after 1 hour, with 10 M NaOH) and heated at 100° C. for 15 minutesto inactivate the enzymes. The final gluten solution was filteredthrough cheesecloth to remove residual large particles. One PEPcapsule-fed animal and one sham capsule-fed animal was sacrificed after45 min and 90 min each, and the small intestinal contents were analyzedfor gluten content via C18 reversed phase HPLC. Chromatograms werenormalized for total protein content in each sample. Top=45 min,Bottom=90 min (green=placebo, blue ═PEP capsule).

As shown in FIGS. 15A and 15B, gluten-derived peptides elute in the20-30 min region. At 45 min as well as 90 min, thepepsin-trypsin-chymotrypsin treated gluten was minimally metabolized inthe sham-fed animals, whereas it appears to be extensively metabolized.Together, these results indicate that enteric-coated PEP capsules cansurvive the gastric environment of the stomach, and catalyze proteolysisof dietary peptides in the small intestine.

Example 11 Detoxification of Gluten by Enzyme Treatment

Prolyl endopeptidases (PEPs) can be administered at the time of a meal,to be released or activated in the upper intestinal lumen where theycomplement the pancreatic proteases by further processing the toxicgliadin peptides in the intestinal lumen, thereby preempting theirinteraction with the intestinal surface.

It was first established that a low-dose, short-term (5-10 g/day for 14days) gluten oral supplement induces fat and carbohydrate malabsorptionwhen given to asymptomatic Celiac Sprue patients on an otherwise glutenfree diet. This protocol was modified to examine the effect of glutenpre-treated with pepsin, trypsin and chymotrypsin (PTC-Gluten) ascompared to PTC-Gluten additionally treated with PEP (PTC-Gluten+PEP).In this study, Celiac patients maintaining a gluten-free diet ingested asupplement of 5 g/day gluten as PTC-Gluten or PTC-Gluten+PEP in anorange-lemon juice vehicle for 14 days in a double-blind crossovermanner. A 6-week washout interval between individual arms of thecrossover trial allowed patients to recover completely from the effects,if any, of the test material in the first arm. The results indicate thatingestion of PEP-treated gluten with its reduced or absent gliadinpeptides does not produce a malabsorptive response in Celiac Patients,and demonstrates that oral supplementation with this peptidase istherapeutic in Celiac Sprue.

Subjects and protocols: This study was approved by the InstitutionalReview Board of the Palo Alto Medical Foundation in Palo Alto, Calif.,and all participants in the study were counseled regarding risks andsigned an informed consent document. 22 patients were recruited who werein symptomatic remission on a gluten-free diet. Two patients dropped outof the study, one near the end of the first stage and the other prior tothe second stage. The 20 patients who completed the study included 7 menand 15 women aged 21 to 78 (mean age 49). The time since diagnosis forthe 20 patients ranged from 3 months to 18 years (mean 6 years).Patients provided copies of their initial small-intestinal biopsypathology report and laboratory reports documenting a history of atleast one positive celiac antibody (gliadin, endomysial ortransglutaminase) to verify a diagnosis of Celiac Sprue. They alsocompleted an entry questionnaire inquiring about their medical history,adherence to the gluten-free diet, and current symptoms. The format ofthe study was a double-blinded crossover in which each patient consumedeither a low daily dose of a gluten supplement (5 g; equivalent ofone-half slice of bread) that was predigested with pepsin, trypsin andchymotrypsin alone (PTC-Gluten) or PTC-Gluten treated with prolylendopeptidase (PTC-Gluten+PEP). The PEP in the latter orange juicemixture was completely inactivated through a heating process followingproteolysis of the gliadin peptides. Each stage consisted of 14 days,separated by a washout period of 6 weeks. Following the 6 week washoutperiod, the patients switched in stage 2 to consuming the other type oforange juice mixture daily for 14 days.

Studied variables: During each stage of the study, patients completed adaily questionnaire inquiring about symptoms and their adherence to thegluten-free diet. Patients documented the presence or absence of 13symptoms, including diarrhea, abdominal bloating, excessive gas passage,abdominal discomfort or pain, nausea, and fatigue by use of a 0-3ordinal scale (none, mild, moderate, and severe) yielding a maximumpossible daily symptom score of 39. Patients also indicated theirperceived adherence to a gluten-free diet using a 0-3 scale with 0, 1,2, and 3 indicating no ingestion of gluten, mild, moderate, and severecontamination, respectively. All laboratory tests were performed byQuest Diagnostics Laboratory in San Juan Capistrano, Calif. A Celiacantibody panel, comprised of serum anti-gliadin IgA and IgG antibodies,anti-tissue transglutaminase IgA and IgG antibodies and a total serumIgA level was obtained at baseline (within 2 weeks of the start of thestudy). The serum anti-gliadin IgA antibodies and anti-tissuetransglutaminase IgA and IgG antibodies were measured by an enzymelinked immunosorbent assay (ELISA) kit (INOVA Diagnostics, Inc., SanDiego, Calif.). The results were obtained by constructing standardcurves with dilutions of a positive reference serum and converted toconcentrations of arbitrary ELISA units (normal <20 EU/ml). Total serumIgA levels were determined by an Integra instrument (Roche) (normal <81mg/dl). A complete blood count with automated differential was obtainedbefore and after each stage. The 25 g D-xylose 5-hour urine excretiontest and the 72-hour quantitative fecal fat test were measured atbaseline and on Day 15. Following an overnight fast and after passingand discarding the urine, patients consumed a 25-gram oral dose ofD-xylose dissolved in 200-300 ml of water and collected all of the urinevoided during the 5 hours after administration of the dose. Patientswere instructed to drink an additional 400-600 ml of water during thefirst two hours of the test to ensure adequate urine flow. A xyloseexcretion of less than 4 g in 5 hours was considered abnormal. For the72 h quantitative fecal fat test, patients were instructed to consume adiet containing 80-100 g fat per day during the collection. The fecalfat excretion was determined by a gravimetric method.

Production of the gluten-containing orange juice mixture: Because it wastechnically cumbersome to work with precise amounts of gluten for use inmaking food products such as bread, muffins, or rolls, we produced anorange juice drink into which an exact amount of cooked wheat glutenflour could be readily incorporated. All components of the Orange JuiceMixture were food grade. Commercially available wheat gluten flour(Bob's Red Mill, Milwaukie Oreg.) was added to 10 L of a 0.01 M HClsolution to achieve a pH of 2.0. Pepsin (6.0 g, Pepsin (P) NF powder,1:10000, American Laboratories, Omaha Nebr.) was added. After incubationat 37° C. for 1 h, the pH was adjusted to 2.0 by addition of 35 ml 1MHCl. After maintenance for an additional 2 h at 37° C., the solution wasneutralized by addition of 35 g of Na₂HPO₄, and the pH was adjusted to7.9 with 10 M NaOH (32.5 ml). Trypsin (T)/Chymotrypsin (C) powder (3.75g) (Enzyme Development Corp., New York, N.Y.; 1000 USP/mg in trypsin,1000 USP/mg in chymotrypsin) was then added, the reaction maintained at37° C. for 2 hours, pH 7.9 (pH re-adjustment to 7.9 after 1 hour, with10 M NaOH) and heated at 100° C. for 15 minutes to inactivate theenzymes. The final PTC-Gluten solution was filtered through cheeseclothto remove residual large particles. Analysis of the protein content(Lowry method) of the filter residue showed no significant loss ofprotein in the residue on the cheesecloth. This treatment of gluten wasshown to yield the final gliadin peptides by analysis on a reverse phaseC-18 HPLC column. Frozen Orange Juice Concentrate (Minute Maid, thawed,2 oz/dose) and Lemon Juice Concentrate (Minute Maid, 0.5 oz/dose) wereadded to the PTC-Gluten solution, mixed, and transferred to plasticbeverage containers (8 oz/5 g gluten). The containers were stored in a−20 C freezer until the day before use. Analysis revealed that thegluten digests were stable by HPLC analysis for at least 60 days.Preparation of PTC-Gluten+PEP was identical to the above protocol,except that the PTC-Gluten was solution was treated with 200 units PEP/ggluten for 1 h prior to heat treatment.

Results:

Baseline Labs: Since the Celiac serum antibody titers may beintermittently abnormal in this disease, even when patients are in fullclinical remission, the antibody levels were not considered to be anexclusion criterion for participation in the study. None of the 20patients who completed the study was IgA deficient. Eight patients (40%)had at least one abnormal antibody level at baseline. Five out of the 20patients had missing baseline xylose urine test results due tolaboratory error. Of the remaining 15 patients, 3 (20%) had an abnormalurine xylose at baseline. One out of the 20 patients had a missingbaseline fecal fat result, again due to laboratory error. Twelve of theremaining 19 patients (63%) had an abnormal baseline fecal fat. None ofthe patients had all 3 baseline tests (an antibody, urine xylose, andfecal fat) abnormal, but 5 patients had 2 abnormal tests (4 with anabnormal antibody and an abnormal fecal fat, 1 with an abnormal antibodyand an abnormal xylose, and 2 with an abnormal fecal fat and an abnormalxylose, Table 5A-B). Therefore, only 3 of the 14 patients with fullbaseline data had a normal antibody panel, normal fecal fat, and normalurine xylose. There was no significant correlation between baseline labvalues and time since diagnosis or perceived dietary adherence in the 2weeks prior to study entry.

TABLE 5a Baseline Labs for patients on a gluten-free diet for <=2 yearsPatient A D E G I L O O T Ttg IgG 8 16 6 7 8 8 5 3 5 (normal <20) TtgIgA 161 19 6 19 13 200 47 15 9 (normal <20) AGA IgA 21 19 7 10 11 91 128 11 (normal <20) Fecal Fat 12 5.1 1.9 22 4.4 13 * 7.6 25 (normal <7g/24 hrs) Urine Xylose 6.7 6.2 6.0 * * 5.9 4.2 2.2 6.2 (normal >4 g/5hrs) Years since diagnosis 0.25 2 0.67 1 2 0.5 2 0.33 1.33

TABLE 5b Baseline Labs for patients on a gluten-free diet for >2 yearsPatient B C F H J K M N P R S U V Ttg IgG 9 8 7 33 10 16 5 3 5 5 5 10 35(normal <20) Ttg IgA 16 43 5 10 119 26 8 11 3 14 8 7 10 (normal <20) AGAIgA 14 11 8 7 40 23 9 11 8 16 17 9 11 (normal <20) Fecal Fat 10 14 4.518 5.3 3.4 8.2 11 11 6.6 15 6 3.9 (normal <7 g/24 hrs) Urine Xylose *4.8 * 5.3 2.2 * 5.5 * 3.0 4.0 6.1 9.9 5.6 (normal >4 g/5 hrs) Yearssince diagnosis 3.25 18 8 10 8 3.17 6.67 11 11 4 17 14.8 2.75

Questionnaire: Participants were asked to maintain a strict gluten-freediet during the study. There was no significant difference in dietaryadherence between the two stages for any of the 20 patients. Eight ofthe 20 patients (40%) reported no perceived episodes ofgluten-contamination in their food intake during either stage. Themajority of the other patients reported ingesting small amounts ofgluten containing foods (mild contamination) for 1-3 days in one or bothstages. Two patients (10%) reported contamination on at least half ofthe days of each stage. Both of these patients had two abnormal baselinetests. In addition, five of the 20 patients (including the two patientsjust mentioned) perceived that they had mild gluten contaminationsometime during the 2 weeks prior to entry into the study while themajority of patients (15 of 20; 75%) reported that they had maintained astrict gluten-free diet during that time frame. Daily symptom scoresranged from 0 to 22, out of a maximum possible score of 39. Mostpatients had relatively few symptoms during either the control or PEPtreatment stage. The average Total Stage Symptom Score (the sum of the14 daily symptom scores for that stage) was 22 (range 0 to 71) for theControl Stage, and 23 (range 4 to 82) for the PEP Stage. There was nocorrelation of symptoms as a function of the gluten preparation.

Gluten Challenge and PEP: To determine whether PEP was effective inavoiding a malabsorptive response, we identified those patients who hada positive control phase, i.e. a positive gluten challenge, based onfecal fat or xylose testing. For patients who had a positive glutenchallenge, the putative avoidance of a malabsorptive response could thenbe determined. A patient was considered to have a positive glutenchallenge based on xylose if the pre-stage and post-stage xylose valuesmet either one of the following criteria: 1) pre-stage urine xylose wasnormal (>4.0 g/5 h) and the post-stage xylose was abnormal (<4.0 g/5 h);or 2) there was at least a 25% decline in the urine xylose frompre-stage to post-stage. A patient was considered to have a positivegluten challenge based on fecal fat if the patient's pre-stage andpost-stage fecal fat values met either one of the following criteria: 1)the pre-stage fecal fat was normal (<7.0 g/24 h) and the post-stagefecal fat was abnormal (>7.0 g/24 h); or 2) if the pre-stage fecal fatwas abnormal, the post-stage fecal fat revealed at least a 25% increasefrom the pre-stage value.

TABLE 6 Results of the Gluten Challenge Based on Urine Xylose Allpatients with Subgroup with normal full xylose (>4 gm/5 hrs) Pre-stagedata (N = 14) Xylose (N = 10) Positive Gluten 8/14 57% 5/10 50%Challenge PEP Effective 4/8  50% 3/5  60%

Fourteen patients had a full set of urine xylose data to analyze. In 5of the remaining 6 patients the baseline urine sample volume waserroneously not recorded, and an additional patient had insufficienturinary output (35 ml for a 5-hour collection)). Using the abovecriteria, we assessed the response to the gluten challenge in the groupof fourteen patients as a whole, and also the subgroup of patients thathad no pre-stage xylose malabsorption (FIG. 2). In the first analysis, 8of the 14 patients (57%) developed xylose malabsorption when challengedwith PTC-Gluten. All 6 of the patients failing to respond to the glutenchallenge had significant (>10 g/24 h) pre-existing fat malabsorption,in contrast to none of the 8 patients who had a positive glutenchallenge. PEP was effective in obviating the development of xylosemalabsorption in the other stage in 4 patients (50%) (Table 5a). In thesubgroup of patients having normal pre-stage (i.e. before both stages)xylose absorption (10 patients), 5 (50%) patients had a positive glutenchallenge (Table 7, FIG. 16). Three of these 5 patients (60%) avoideddeveloping xylose malabsorption when taking the gluten pre-treated withPEP.

TABLE 7a Urine Xyloses for Patients with a Positive Xylose GlutenChallenge No pep pep Patient Pre-Stage Post-Stage Pre-Stage Post-Stage D9.4 4.4 6.2 7.2 E 6.0 1.3 3.4 6.1 J 2.2 0.8 4.3 2.5 M 5.5 3.2 7.8 3.6 O4.2 3.1 4.3 4.1 Q 2.4 0.8 2.2 1.6 U 9.9 4.2 7.3 1.4 V 5.6 3.2 6.2 6.0

The stool for fecal fat analysis was lost in one of the 20 patients, andtwo patients had an incomplete stool collection (<150 g/24 h) for one ofthe four stool collections. These patients were excluded from subsequentanalysis. Of the remaining 17 patients, 7 had a positive glutenchallenge based on the fecal fat test (Tables 8, 9). In this group ofgluten-responsive patients, ingestion of PEP-treated gluten avoided thedevelopment of fat malabsorption in the treatment stage in 5 of the 7(71%). Notably, a minority of these asymptomatic Celiac patients inapparent remission (3 of 17 patients (18%)) had completely normalpre-stage fat absorption, but most of these had very a very mildincrease in the fecal fat excretion. Analysis of the larger subgroup of8 patients (47%) with either normal fat absorption or only mildsteatorrhea (fecal fat up to 10 g/24 h) (Table 8, 9), revealed that fourhad a positive gluten challenge. All of these individuals (100%)absorbed fat normally after ingesting the PEP-treated gluten.

TABLE 8 Results of the Gluten Challenge Based on Fecal Fat Testing Allpatients with full Subgroup with normal to Fecal Fat data (N = 17) MildSteatorrhea (N = 8) Positive Gluten 7/17 41% 4/8  50% Challenge PEPEffective 5/7  71% 4/4 100%

TABLE 9 Fecal Fats of Patients with a Positive Fecal Fat GlutenChallenge No pep pep Patient Pre-Stage Post-Stage Pre-Stage Post-Stage B4.3 12 10 5.7 M 8.2 11 7.7 4.6 P 4.9 12 11 5.8 S 15 24 8.2 25 T 16 22 2515 U 6 11 7.8 5.6 V 3.9 3.4 3.4 5.4

In this double-blind trial, symptoms were not distinguishable during thecontrol (PTC-Gluten) versus the PEP-digested (PTC-Gluten+PEP) stage,despite a high prevalence of fat malabsorption (66%) and carbohydrate(21%) in the Celiac patients in remission at baseline, before eithertype of pre-treated gluten was consumed. The fecal fat is a moresensitive test for documenting intestinal dysfunction than the xyloseurine test, and this is consistent with the finding that only one-thirdof patients with baseline fat malabsorption were found to have anabnormal xylose test. Nevertheless, the absorption of xylose requires nopancreatic luminal enzymes and hence even a borderline abnormal valueindicates the reason for increased stool fat output is due to an entericrather than a pancreatic lesion.

Celiac serum antibody titers (gliadin, endomysial, transglutaminase)have been found to correlate closely with the histological findings inuntreated Celiac Sprue, and are frequently used to monitor adherence toa gluten-exclusion diet. However, we had found that 21 days of low-dose(5-10 Gm per day) oral gluten supplementation does not convert theseantibody studies to the abnormal range. The current study revealed that7 patients (#2, 7, 12, 13, 15, 16, 17, 18; see tables) had elevatedtransglutaminase titers at baseline, 6 of these being IgA typeantibodies and one IgG. All of these patients also had malabsorption.Four of these same patients were positive for anti-gliadin antibodies.However, most striking was the fact that all antibodies were negative in8 other patients who had malabsorption of fat or carbohydrate. Itappears that intestinal absorptive tests may be superior to the serumantibody tests in monitoring the intestinal function in Celiac Sprue.

The finding that two-thirds of Celiac individuals in clinical remissionhave fat malabsorption and one of five have carbohydrate malabsorptionsuggests strongly that intestinal dysfunction due to continuingintestinal injury occurs commonly in the disease, despite lack ofsymptoms and maintenance of a gluten-exclusion diet. This emphasizes theneed for identifying incremental long-term therapy for Celiac Sprue inaddition to the conventional dietary gluten exclusion. Celiac Spruepatients with chronic fat or carbohydrate malabsorption could be treatedwith oral enzyme therapy in order to improve their malabsorptivesymptoms with the longer-term goal of reducing the relatively highprevalence of osteopenia and iron deficiency anemia. An endoscopicbiopsy would also be indicated whenever malabsorption is identified, andcould be a useful tool to monitor therapeutic efficacy of the oralenzyme.

In those who had normal intestinal function, as evidenced by the absenceof malabsorption in the pre-test stage and who then mounted amalabsorptive response to PTC-Gluten ingestion, no malabsorption wasobserved in the majority of these gluten-responsive Celiac patients whenthe gliadin peptides from PTC-Gluten were further processed prior toingestion by PEP treatment (PTC-Gluten+PEP). This indicates that thepre-treatment of dietary gluten peptides with PEP results in appreciableablation of their toxic properties for the Celiac small intestine. Thefact that PEP treated gluten does not induce malabsorption demonstratesstrongly that this peptidase is sufficient to eliminate the bulk ofinjurious gliadin in food sources of gluten, for example as an oralpeptidase supplement as supportive therapy in patients with CeliacSprue. The need for additional therapy in Celiac Sprue is even moreessential in view of the finding that many Celiac patients in fullclinical remission have continuing sub-clinical malabsorption,undoubtedly due to continuing intestinal damage, despite theirmaintenance of dietary gluten exclusion.

Based on the above results, the clinical trials may be varied asfollows. Since not all of the Celiac Sprue patents in clinical remissionresponded to a 5 g gluten dose with fat and carbohydrate malabsorptionin this study, future studies may increase the gluten dose to 10 g/day.In addition, the radiotelemetry video capsule may provide moreinformation for assessment of overall small intestinal structure.Finally, an oligosaccharide permeability test as an additionalassessment of intestinal integrity may be desirable.

All publications, patents, and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication, patent, or patent application were specifically andindividually indicated to be incorporated by reference.

The present invention has been described in terms of particularembodiments found or proposed by the inventor to comprise preferredmodes for the practice of the invention. It will be appreciated by thoseof skill in the art that, in light of the present disclosure, numerousmodifications and changes can be made in the particular embodimentsexemplified without departing from the intended scope of the invention.Moreover, due to biological functional equivalency considerations,changes can be made in methods, structures, and compounds withoutaffecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

1. A method of treating Celiac Sprue and/or dermatitis herpetiformis, the method comprising: administering to a patient an effective dose of a glutenase; wherein said glutenase attenuates gluten toxicity in said patient and is administered in two enzyme doses, one dose containing a prolyl endopeptidase (PEP), and the other dose containing a glutamine endoprotease.
 2. (canceled)
 3. The method according to claim 1, wherein said prolyl endopeptidase is Flavobacterium meningosepticum PEP.
 4. The method according to claim 1, wherein said prolyl endopeptidase is Myxococcus xanthus PEP.
 5. The method according to claim 1, wherein said prolyl endopeptidase is Sphingomonas capsulata PEP.
 6. The method according to claim 1, wherein said prolyl endopeptidase is Lactobacillus helveticus PEP.
 7. The method according to claim 1, wherein said prolyl endopeptidase is Penicillium citrinum PEP.
 8. (canceled)
 9. The method according to claim 1, wherein said glutamine specific protease is Hordeum vulgare endoprotease. 10-11. (canceled)
 12. The method according to claim 1, wherein said glutenase is formulated with a pharmaceutically acceptable excipient.
 13. (canceled)
 14. The method according to claim 1, wherein said glutenase is stable to acid conditions.
 15. The method according to claim 1, wherein said glutenase is administered orally.
 16. The method according to claim 9, wherein said glutenase is contained in a formulation that comprises an enteric coating.
 17. A formulation for use in treatment of Celiac Sprue and/or dermatitis herpetiformis, comprising: an effective dose of glutenase and a pharmaceutically acceptable excipient wherein said glutenase is formulated in two enzyme doses, one dose containing a prolyl endopeptidase (PEP), and the other dose containing a glutamine endoprotease.
 18. (canceled)
 19. The formulation according to claim 17, wherein said prolyl endopeptidase is Flavobacterium meningosepticum PEP.
 20. The formulation according to claim 17, wherein said prolyl endopeptidase is Myxococcus xanthus PEP.
 21. The formulation according to claim 17, wherein said prolyl endopeptidase is Sphingomonas capsulata PEP.
 22. The formulation according to claim 17, wherein said prolyl endopeptidase is Lactobacillus helveticus PEP.
 23. The formulation according to claim 17, wherein said prolyl endopeptidase is Penicillium citrinum PEP.
 24. (canceled)
 25. The formulation according to claim 17, wherein said glutamine specific protease is Hordeum vulgare endoprotease. 26-27. (canceled)
 28. The formulation according to claim 17, wherein said glutenase is stable to acid conditions.
 29. The formulation according to claim 17, wherein said formulation is suitable for oral administration.
 30. The formulation according to claim 17, wherein said formulation comprises an enteric coating. 31-35. (canceled)
 36. A composition, comprising: isolated Myxococcus xanthus PEP. 